Stabilized protein crystals formulations containing them and methods of making them

ABSTRACT

This invention relates to methods for the stabilization, storage and delivery of biologically active macromolecules, such as proteins, peptides and nucleic acids. In particular, this invention relates to protein or nucleic acid crystals, formulations and compositions comprising them. Methods are provided for the crystallization of proteins and nucleic acids and for the preparation of stabilized protein or nucleic acid crystals for use in dry or slurry formulations. The present invention is further directed to encapsulating proteins, glycoproteins, enzymes, antibodies, hormones and peptide crystals or crystal formulations into compositions for biological delivery to humans and animals. According to this invention, protein crystals or crystal formulations are encapsulated within a matrix comprising a polymeric carrier to form a composition. The formulations and compositions enhance preservation of the native biologically active tertiary structure of the proteins and create a reservoir which can slowly release active protein where and when it is needed. Methods are provided preparing stabilized formulations using pharmaceutical ingredients or excipients and optionally encapsulating them in a polymeric carrier to produce compositions and using such protein crystal formulations and compositions for biomedical applications, including delivery of therapeutic proteins and vaccines. Additional uses for the protein crystal formulations and compositions of this invention involve protein delivery in human food, agricultural feeds, veterinary compositions, diagnostics, cosmetics and personal care compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of co-pending InternationalApplication PCT/US99/09099, filed Apr. 27, 1999, which claims benefitunder 35 U.S.C. § 119(e) of U.S. provisional application No. 60/083,148filed on Apr. 27, 1998. This application is also a continuation-in-partof U.S. application 09/224,475, filed on Dec. 31, 1998, which nowabandoned, which claims benefit under 35 U.S.C. § 119(e) of U.S.provisional application No. 60/070,274, filed on Dec. 31, 1997.

TECHNICAL FIELD OF THE INVENTION

This invention relates to methods for the stabilization, storage anddelivery of biologically active macromolecules, such as proteins,peptides and nucleic acids. In particular, this invention relates toprotein or nucleic acid crystals, formulations and compositionscomprising them. Methods are provided for the crystallization ofproteins and nucleic acids and for the preparation of stabilized proteinor nucleic acid crystals for use in dry or slurry formulations. Thecrystals, crystal formulations and compositions of this invention can bereconstituted with a diluent for the parenteral administration ofbiologically active macromolecular components.

The methods of this invention are useful for preparing crystals of“naked” DNA and RNA sequences that code for therapeutic or immunogenicproteins and can be administered parenterally. The dissolving DNA andRNA molecules, subsequently taken up by the cells and used to expressthe protein with the proper glycosylation pattern, can be eithertherapeutic or immunogenic. Alternatively, the present invention isuseful for preparing crystals, crystal formulations and compositions ofsense and antisense polynucleotides of RNA or DNA.

The present invention is further directed to encapsulating proteins,glycoproteins, enzymes, antibodies, hormones and peptide crystals orcrystal formulations into compositions for biological delivery to humansand animals. According to this invention, protein crystals or crystalformulations are encapsulated within a matrix comprising a polymericcarrier to form a composition. The formulations and compositions enhancepreservation of the native biologically active tertiary structure of theproteins and create a reservoir which can slowly release active proteinwhere and when it is needed. Such polymeric carriers includebiocompatible and biodegradable polymers. The biologically activeprotein is subsequently released in a controlled manner over a period oftime, as determined by the particular encapsulation technique, polymerformulation, crystal geometry, crystal solubility, crystal crosslinkingand formulation conditions used. Methods are provided for crystallizingproteins, preparing stabilized formulations using pharmaceuticalingredients or excipients and optionally encapsulating them in apolymeric carrier to produce compositions and using such protein crystalformulations and compositions for biomedical applications, includingdelivery of therapeutic proteins and vaccines. Additional uses for theprotein crystal formulations and compositions of this invention involveprotein delivery in human food, agricultural feeds, veterinarycompositions, diagnostics, cosmetics and personal care compositions.

BACKGROUND OF THE INVENTION

Proteins are used in a wide range of applications in the fields ofpharmaceuticals, veterinary products, cosmetics and other consumerproducts, foods, feeds, diagnostics, industrial chemistry anddecontamination. At times, such uses have been limited by constraintsinherent in proteins themselves or imposed by the environment or mediain which they are used. Such constraints may result in poor stability ofthe proteins, variability of performance or high cost.

It is imperative that the higher order three-dimensional architecture ortertiary structure of a protein be preserved until such time that theindividual protein molecules are required to perform their uniquefunction. To date, a limiting factor for use of proteins, particularlyin therapeutic regimens, remains the sensitivity of protein structure tochemical and physical denaturation encountered during delivery.

Various approaches have been employed to overcome these barriers.However, these approaches often incur either loss of protein activity orthe additional expense of protein stabilizing carriers or formulations.

One approach to overcoming barriers to the widespread use of proteins iscrosslinked enzyme crystal (“CLEC™”) technology [N. L. St. Clair and M.A. Navia, J. Am. Chem. Soc., 114, pp. 4314-16 (1992)]. See also PCTpatent application PCT/US91/05415. Crosslinked enzyme crystals retaintheir activity in environments that are normally incompatible withenzyme function. Such environments include prolonged exposure toproteases, organic solvents, high temperature or extremes of pH. In suchenvironments, crosslinked enzyme crystals remain insoluble, stable andactive.

Despite recent progress in protein technology generally, two problemswhich are discussed below continue to limit the use of biologicalmacromolecules in industry and medicine. The first problem relates tomolecular stability and sensitivity of higher order tertiary structuresto chemical and physical denaturation during manufacturing and storage.Second, the field of biological delivery of therapeutic proteinsrequires that vehicles be provided which release native proteins, suchas proteins, glycoproteins, enzymes, antibodies, hormones, nucleic acidsand peptides at a rate that is consistent with the needs of theparticular patient or the disease process.

Macromolecule Stability

Numerous factors differentiate biological macromolecules fromconventional chemical entities, such as for example, their size,conformation and amphiphilic nature. Macromolecules are not onlysusceptible to chemical, but also physical degradation. They aresensitive to a variety of environmental factors, such as temperature,oxidizing agents, pH, freezing, shaking and shear stress [Cholewinski,M., Luckel, B. and Horn, H., Acta Helv., 71, 405 (1996)]. In consideringa macromolecule for drug development, stability factors must beconsidered when choosing a production process.

Maintenance of biological activity during the development andmanufacture of pharmaceutical products depends on the inherent stabilityof the macromolecule, as well as the stabilization techniques employed.A range of protein stabilization techniques exist; including:

a) Addition of chemical “stabilizers” to the aqueous solution orsuspension of protein. For example, U.S. Pat. Nos. 4,297,344 disclosesstabilization of coagulation factors II and VIII, antithrombin III andplasminogen against heat by adding selected amino acids. U.S. Pat. No.4,783,441 discloses a method for stabilizing proteins by addingsurface-active substances. U.S. Pat. No. 4,812,557 discloses a methodfor stabilizing interleukin-2 using human serum albumin. The drawback ofsuch methods is that each formulation is specific to the protein ofinterest and requires significant development efforts.

b) Freeze/thaw methods in which the preparation is mixed with acryoprotectant and stored at very low temperatures. However, not allproteins will survive a freeze/thaw cycle.

c) Cold storage with cryoprotectant additive, normally glycerol.

d) Storage in the glass form, as described in U.S. Pat. No. 5,098,893.In this case, proteins are dissolved in water-soluble or water-swellablesubstances which are in amorphous or glassy state.

e) The most widely used method for the stabilization of proteins isfreeze-drying or lyophilization [Carpenter, J. F., Pical, M. J., Chang,B. S. and Randolph, T. W., Pharm. Res., 14:(8) 969 (1997)]. Wheneversufficient protein stability cannot be achieved in aqueous solution,lyophilization provides the most viable alternative. One disadvantage oflyophilization is that it requires sophisticated processing, is timeconsuming and expensive [Carpenter, J. F., Pical, M. J., Chang, B. S.and Randolph, T. W., Pharm. Res., 14:(8) 969 (1997) and literature citedtherein]. In addition, if lyophilization is not carried out carefully,most preparations are at least partially denatured by the freezing anddehydration steps of the technique. The result is frequentlyirreversible aggregation of a portion of protein molecules, rendering aformulation unacceptable for parenteral administration.

The vast majority of protein formulations produced by theabove-described techniques require cold storage, sometimes as low as−20° C. Exposure to elevated temperatures during shipping or storage canresult in significant activity losses. Thus, storage at elevated, oreven ambient temperatures, is not possible for many proteins.

Proteins, peptides and nucleic acids are increasingly employed in thepharmaceutical, diagnostic, food, cosmetic, detergent and researchindustries. There is a great need for alternative stabilizationprocedures, which are fast, inexpensive and applicable to a broad rangeof biological macromolecules. In particular, stabilization proceduresare needed that do not rely on the excessive use of excipients, whichcan interfere with the functions of those biological macromolecules.

The stability of small molecule crystalline drugs is such that they canwithstand extreme forces during the formulation process (see U.S. Pat.No. 5,510,118). Forces associated with milling nanoparticles ofcrystalline material of relatively insoluble drugs include: shearstress, turbulent flow, high impact collisions, cavitation and grinding.Small molecular crystalline compounds have been recognized as being muchmore stable toward chemical degradation than the corresponding amorphoussolid [Pical, M. J., Lukes, A. L., Lang, J. E. and Gaines, J. Pharm.Sci., 67, 767 (1978)]. Unfortunately, crystals of macromolecules, suchas proteins and nucleic acids, present additional problems anddifficulties not associated with small molecules.

For most of this century, science and medicine have tried to solve theproblem of providing insulin in a useful form to diabetics. Attemptshave been made to solve some of the problems of stability and biologicaldelivery of that protein. For example, U.S. Pat. No. 5,506,203 describesthe use of amorphous insulin combined with an absorbtion enhancer. Thesolid state insulin was exclusively amorphous material, as shown by apolarized light microscope.

Jensen et al. co-precipitated insulin with an absorbtion enhancer foruse in respiratory tract delivery of insulin (See PCT patent applicationWo 98/42368). Here, the absorbtion enhancer was desribed as asurfactant, such as a salt of a fatty acid or a bile salt. Insulincrystals of less than 10 micrometers in diameter and lacking zinc wereproduced by S. Havelund (See PCT patent application Wo 98/42749).Similarly, crystals were also produced in the presence of surfactants toenhance pulmonary administration.

To date, those of skill in the art recognize that the greatly enhancedstability of the crystalline state observed for small molecules does nottranslate to biological macromolecules [Pical, M. J. and Rigsbee, D. R.,Pharm. Res., 14:1379 (1997)]. For example, aqueous suspensions ofcrystalline insulin are only slightly more stable (to the degree of afactor of two) than corresponding suspensions of amorphous phase[Brange, J., Langkjaer, L., Havelund, S. and Volund, A., Pharm. Res.,9:715 (1992)]. In the solid state, lyophilized amorphous insulin is farmore stable than lyophilized crystalline insulin under all conditionsinvestigated [Pical, M. J. and Rigsbee, D. R., Pharm. Res., 14:1379(1997)].

Until now, formulations of crystalline proteins have been available onlyfor very small proteins, e.g. proteins with molecular weights of lessthan 10,000 Daltons. Molecular weight has profound effect on allproperties of macromolecules, including their macromolecular volume,hydration, viscosity, diffusion, mobility and stability. [Cantor, C. Rand Schimmel, P. R, Biophysical Chemistry, W. H. Freeman and Co., NewYork, 1980].

SUMMARY OF THE INVENTION

We have found, surprisingly, that biological macromolecules which arenot stable when held in solution at ambient or elevated temperatures cannevertheless be successfully stored in dry form for long periods of timeat such temperatures in crystalline form. As a practical matter, fiveaspects of this discovery are particularly advantageous.

First, crystallinity of stored materials is very important, since largescale crystallization can be introduced as a final purification stepand/or concentration step in clinical manufacturing processes, such asthose for manufacturing therapeutics and vaccines. Moreover, large scalecrystallization can replace some of the purification steps in themanufacturing process. For example, protein crystallization canstreamline the production of protein formulations making it moreaffordable.

Second, macromolecular interactions which occur in solution areprevented or severely reduced in the crystalline state, due toconsiderable reduction of all reaction rates. Thus, the crystallinestate is uniquely suited to the storage of mixtures of biologicalmacromolecules.

Third, solid crystalline preparations can be easily reconstituted togenerate ready to use parenteral formulations having very high proteinconcentration. Such protein concentrations are considered to beparticularly useful where the formulation is intended for subcutaneousadministration. (See PCT patent application Wo 97/04801). Forsubcutaneous administration, injection volumes of 1.5 ml or less arewell tolerated. Thus, for proteins that are dosed at 1 mg/kg on a weeklybasis a protein concentration of at least 50 mg/ml is required and100-200 mg/ml is preferred. These concentrations are difficult toachieve in liquid formulations, due to the aggregation problems. Theycan easily be achieved in the crystalline formulations of thisinvention.

Fourth, protein crystals also constitute a particularly advantageousform for pharmaceutical dosage preparation. The crystals may be used asa basis for slow release formulations in vivo. As those of skill in theart will appreciate, particle size is of importance for the dissolutionof crystals and release of activity. It is also known that the rate ofrelease is more predictable if the crystals have substantially uniformparticle size and do not contain amorphous precipitate (see Europeanpatent 0 265 214). Thus, protein crystals may be advantageously used(see PCT patent application WO 96/40049), on implantable devices.Implant reservoirs are generally on the order of 25-250 μl. With thisvolume restriction, a formulation of high concentration (greater than10%) and a minimum amount of suspension vehicle is preferred. Proteincrystals of this invention may be easily formulated in non-aqueoussuspensions in such high concentrations.

Fifth, another advantage of crystals is that certain variables can bemanipulated to modulate the release of macromolecules over time. Forexample, crystal size, shape, formulation with excipients that effectdissolution, crosslinking, level of crosslinking and encapsulation intoa polymer matrix can all be manipulated to produce delivery vehicles forbiological molecules.

The present invention overcomes the above-described obstacles byemploying the most stable form of an active protein, the crystallineform and either (1) adding ingredients or excipients where necessary tostabilize dried crystals or (2) encapsulating the protein crystals orcrystal formulations within a polymeric carrier to produce a compositionthat contains each crystal and subsequently allows the release of activeprotein molecules. Any form of protein, including glycoproteins,antibodies, enzymes, hormones or peptides, may be crystallized andstabilized or encapsulated into compositions according to the methods ofthis invention. In addition, the nucleic acids coding for such proteinsmay be similarly treated.

The crystal(s) may be encapsulated using a variety of polymeric carriershaving unique properties suitable for delivery to different and specificenvironments or for effecting specific functions. The rate ofdissolution of the compositions and, therefore, delivery of the activeprotein can be modulated by varying crystal size, polymer composition,polymer crosslinking, crystal crosslinking, polymer thickness, polymerhydrophobicity, polymer crystallinity or polymer solubility.

The addition of ingredients or excipients to the crystals of the presentinvention or the encapsulation of protein crystals or crystalformulations results in further stabilization of the proteinconstituent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the relative stability of the following molecular statesof Candida rugosa lipase: crosslinked amorphous, liquid, crystalline in20% organic solvent, crosslinked crystalline in 20% organic solvent,crosslinked crystalline without organic solvent (“Xlinke ppt” denotescrosslinked precipitate).

FIG. 2 depicts the specific activity of soluble lipase over time at 40°C.

FIG. 3 depicts the shelf stabilities of lipase crystal formulationsdried by method 1 at 40° C. and 75% humidity.

FIG. 4 depicts the shelf stabilities of lipase crystal formulationsdried by method 4 at 40° C. and 75% humidity.

FIG. 5 depicts lipase crystals formulated with polyethylene oxide atinitial time 0.

FIG. 6 depicts lipase crystals formulated with polyethylene oxide afterincubation for 129 days at 40° C. and 75% humidity.

FIG. 7 depicts human serum albumin crystals formulated with gelatin atinitial time 0.

FIG. 8 depicts human serum albumin crystals formulated with gelatinafter incubation for 4 days at 50° C.

FIG. 9 depicts the specific activity of soluble Penicillin acylase overtime at 55° C.

FIG. 10 depicts the shelf stabilities of various dried Penicillinacylase crystal formulations at 55° C.

FIG. 11 depicts the specific activity of soluble glucose oxidase overtime.

FIG. 12 depicts the shelf stabilities of various dried glucose oxidasecrystal formulations at 50° C.

FIG. 13 depicts glucose oxidase crystals formulated with lactitol atinitial time 0.

FIG. 14 depicts glucose oxidase crystals formulated with lactitol afterincubation for 13 days at 50° C.

FIG. 15 depicts glucose oxidase crystals formulated with trehalose atinitial time 0.

FIG. 16 depicts glucose oxidase crystals formulated with trehalose afterincubation for 13 days at 50° C.

FIG. 17 depicts encapsulated crosslinked enzyme crystals of lipase fromCandida rugosa.

FIG. 18 depicts encapsulated uncrosslinked enzyme crystals of lipasefrom Candida rugosa.

FIG. 19 depicts encapsulated crosslinked enzyme crystals of Penicillinacylase from Escherichia coli.

FIG. 20 depicts encapsulated uncrosslinked enzyme crystals of Penicillinacylase from Escherichia coli.

FIG. 21 depicts encapsulated crosslinked enzyme crystals of glucoseoxidase from Aspergillus niger.

FIG. 22 depicts encapsulated uncrosslinked enzyme crystals of glucoseoxidase from Aspergillus niger.

FIG. 23 depicts an encapsulated aqueous slurry of uncrosslinked enzymecrystals of lipase from Pseudomonas cepacia.

FIG. 24 depicts uncrosslinked enzyme crystals of lipase from Pseudomonascepacia.

DETAILED DESCRIPTION OF THE INVENTION

In order that the invention herein described may be more fullyunderstood, the following detailed description is set forth. In thedescription, the following terms are employed:

Amorphous solid—a non-crystalline solid form of protein, sometimesreferred to as amorphous precipitate, which has no molecular latticestructure characteristic of the crystalline solid state.

Anti-sense polynucleotides—RNA or DNA which codes for RNA, which iscomplementary to the mRNA of a gene whose expression is intended to beinhibited.

Aqueous-organic solvent mixture—a mixture comprising n% organic solvent,where n is between 1 and 99 and m% aqueous, where m is 100-n.

Biocompatible polymers—polymers that are non-antigenic (when not used asan adjuvant), non-carcinogenic, non-toxic and which are not otherwiseinherently incompatible with living organisms. Examples include: poly(acrylic acid), poly (cyanoacrylates), poly (amino acids), poly(anhydrides), poly (depsipeptide), poly (esters) such as poly (lacticacid) or PLA, poly (lactic-co-glycolic acid) or PLGA, poly(β-hydroxybutryate), poly (caprolactone) and poly (dioxanone); poly(ethylene glycol), poly ((hydroxypropyl)methacrylamide, poly[(organo)phosphazene], poly (ortho esters), poly (vinyl alcohol), poly(vinylpyrrolidone), maleic anhydride-alkyl vinyl ether copolymers,pluronic polyols, albumin, alginate, cellulose and cellulosederivatives, collagen, fibrin, gelatin, hyaluronic acid,oligosaccarides, glycaminoglycans, sulfated polysaccarides, blends andcopolymers thereof.

Biodearadable Polymers—polymers that degrade by hydrolysis orsolubilization. Degradation can be heterogenous—occurring primarily atthe particle surface, or homogenous—degrading evenly throughout thepolymer matrix, or a combination of such processes.

Biological macromolecule—biological polymers such as proteins,deoxyribonucleic acids (DNA) and ribonucleic acids (RNA). For thepurposes of this application, biological macromolecules are alsoreferred to as macromolecules.

Change in chemical composition—any change in the chemical components ofthe environment surrounding a protein or nucleic acid crystal or crystalformulation that affects the stability or rate of dissolution of thecrystal component.

Change in shear force—any change in factors of the environmentsurrounding a protein or nucleic acid crystal or crystal formulationunder conditions of use, such as, changes in mechanical pressure, bothpositive and negative, revolution stirring, centrifugation, tumbling,mechanical agitation and filtration pumping.

Composition—either uncrosslinked protein crystals, crosslinked proteincrystals, nucleic acid crystals or formulations containing them, whichhave been encapsulated within a polymeric carrier to form coatedparticles. As used herein, composition always refers to encapsulatedcrystals or formulations.

Controlled dissolution—dissolution of a protein or nucleic acid crystalor crystal formulation or release of the crystalline constituent of saidformulation that is controlled by a factor selected from the groupconsisting of the following: the surface area of said crystal; the sizeof said crystal; the shape of said crystal, the concentration ofexcipient component; the number and nature of excipient components; themolecular weight of the excipient components and combinations thereof.

Co-polymer—a polymer made with more than one monomer species.

Crystal—one form of the solid state of matter, which is distinct from asecond form—the amorphous solid state. Crystals display characteristicfeatures including a lattice structure, characteristic shapes andoptical properties such as refractive index. A crystal consists of atomsarranged in a pattern that repeats periodically in three dimensions (C.S. Barrett, Structure of Metals, 2nd ed., McGraw-Hill, New York, 1952,p.1). The crystals of the present invention may be protein,glycoprotein, peptide, antibodies, therapeutic proteins, or DNA or RNAcoding for such proteins.

Drying of Protein or Nucleic Acid Crystals—removal of water, organicsolvent or liquid polymer by means including drying with N₂, air orinert gases, vacuum oven drying, lyophilization, washing with a volatileorganic solvent followed by evaporation of the solvent, or evaporationin a fume hood. Typically, drying is achieved when the crystals become afree flowing powder. Drying may be carried out by passing a stream ofgas over wet crystals. The gas may be selected from the group consistingof: nitrogen, argon, helium, carbon dioxide, air or combinationsthereof.

Effective amount—an amount of a protein or nucleic acid crystal orcrystal formulation or composition of this invention which is effectiveto treat, immunize, boost, protect, repair or detoxify the subject orarea to which it is administered over some period of time.

Emulsifier—a surface active agent which reduces interfacial tensionbetween polymer coated protein crystals and a solution.

Formulations or (Protein or nucleic acid crystal formulations)—acombination of the protein or nucleic acid crystals of this inventionand one or more ingredients or excipients, including sugars andbiocompatible polymers. Examples of excipients are described in theHandbook of Pharmaceutical Excipients, published jointly by the AmericanPharmaceutical Association and the Pharmaceutical Society of GreatBritian. For the purposes of this application, “formulations” include“crystal formulations”. Furthermore, “formulations” include “protein ornucleic acid crystal formulations”.

Formulations for decontamination—formulations selected from the groupconsisting of: formulations for decontamination of chemical wastes,herbicides, insecticides, pesticides and environmental hazards.

Gene therapy—therapy using formulations and/or compositions of DNAcoding for a protein which is defective, missing, or insufficientlyexpressed in an individual. The crystals are injected intonon-proliferating tissue where the DNA is taken up into the cells andexpressed for a period of one to six months. The expressed proteinserves to temporarily replace or supplement the endogenous protein. Genetherapy can also serve to inhibit gene expression by providingtransgenes with the gene orientation reversed relative to the promoterso that antisense mRNA is produced in vivo.

Glycoprotein—a protein or peptide covalently linked to a carbohydrate.The carbohydrate may be monomeric or composed of oligosaccharides.

Homo-polymer—a polymer made with a single monomer species.

Immunotherapeutic—a protein derived from a tumor cell, virus or bacteriahaving a protein activity of inducing protective immunity to said tumorcell, virus, or bacteria. An immunotherapeutic may be administereddirectly—as a protein or indirectly—by injecting DNA or RNA which codesfor the protein.

Immunotherapeutics may also be protein or glycoprotein cytokines orimmune cell co-stimulatory molecules which stimulate the immune systemto reduce or eliminate said tumor cell, virus or bacteria.

Liquid Polymer—pure liquid phase synthetic polymers, such aspoly-ethylene glycol (PEG), in the absence of aqueous or organicsolvents.

Macromolecules—proteins, glycoproteins, peptides, therapeutic proteins,DNA or RNA molecules.

Method of Administration—protein or nucleic acid crystals or crystalformulations or compositions may be appropriate for a variety of modesof administration. These may include oral, parenteral, subcutaneous,intravenous, pulmonary, intralesional, or topical administration.Alternatively, nucleic acid crystals may be covalently attached to goldparticles, or other carrier beads, for delivery to non-proliferatingtissues such as muscles with a “DNA gun”.

Naked DNA—a nonreplicating, nonintegrating polynucleotide which codesfor a vaccine antigen, therapeutic protein, or immunotherapeuticprotein, which may be operatively linked to a promoter and inserted intoa replication competent plasmid. The DNA is free from association withtransfection facilitating proteins, viral particles, liposomalformulations, lipids and calcium phosphate precipitating agents.

Naked DNA vaccine—crystals of DNA coding for a vaccine antigen or avaccine antigen and an immunotherapeutic. The vaccine is injected intonon-proliferating tissue where the DNA is taken up into the cells andexpressed for a period of one to six months. The nucleic acid crystalsmay be covalently linked to gold particles to aid in delivery to thesite of administration.

Organic solvents—any solvent of non-aqueous origin, including liquidpolymers and mixtures thereof. Organic solvents suitable for the presentinvention include: acetone, methyl alcohol, methyl isobutyl ketone,chloroform, 1-propanol, isopropanol, 2-propanol, acetonitrile,1-butanol, 2-butanol, ethyl alcohol, cyclohexane, dioxane, ethylacetate, dimethylformamide, dichloroethane, hexane, isooctane, methylenechloride, tert-butyl alchohol, toluene, carbon tetrachloride, orcombinations thereof

Peptide—a polypeptide of small to intermediate molecular weight, usually3 to 35 amino acid residues and frequently but not necessarilyrepresenting a fragment of a larger protein.

Pharmaceutically effective amount—an amount of a protein or nucleic acidcrystal or crystal formulation or composition which is effective totreat a condition in an living organism to whom it is administered oversome period of time.

Ingredients—any excipient or excipients, including pharmaceuticalingredients or excipients. Excipients include, for example, thefollowing:

Acidifying Agents

acetic acid, glacial acetic acid, citric acid, fumaric acid,hydrochloric acid, diluted hydrochloric acid, malic acid, nitric acid,phosphoric acid, diluted phosphoric acid, sulfuric acid, tartaric acid

Aerosol Propellants

butane, dichlorodifluoromethane, dichlorotetrafluoroethane, isobutane,propane, trichloromonofluoromethane

Air Displacements

carbon dioxide, nitrogen

Alcohol Denaturants

denatonium benzoate, methyl isobutyl ketone, sucrose octacetate

Alkalizing Agents

strong ammonia solution, ammonium carbonate, diethanolamine,diisopropanolamine, potassium hydroxide, sodium bicarbonate, sodiumborate, sodium carbonate, sodium hydroxide, trolamine

Anticaking Agents (See Glidant)

Antifoaming Agents

dimethicone, simethicone

Antimicrobial Preservatives

benzalkonium chloride, benzalkonium chloride solution, benzelthoniumchloride, benzoic acid, benzyl alcohol, butylparaben, cetylpyridiniumchloride, chlorobutanol, chlorocresol, cresol, dehydroacetic acid,ethylparaben, methylparaben, methylparaben sodium, phenol, phenylethylalcohol, phenylmercuric acetate, phenylmercuric nitrate, potassiumbenzoate, potassium sorbate, propylparaben, propylparaben sodium, sodiumbenzoate, sodium dehydroacetate, sodium propionate, sorbic acid,thimerosal, thymol

Antioxidants

ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylatedhydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate,sodium formaldehyde sulfoxylate, sodium metabisulfite, sodiumthiosulfate, sufur dioxide, tocopherol, tocopherols excipient

Buffering Agents

acetic acid, ammonium carbonate, ammonium phosphate, boric acid, citricacid, lactic acid, phosphoric acid, potassium citrate, potassiummetaphosphate, potassium phosphate monobasic, sodium acetate, sodiumcitrate, sodium lactate solution, dibasic sodium phosphate, monobasicsodium phosphate

Capsule Lubricants (See Tablet and Capsule Lubricant)

Chelating Agents

edetate disodium, ethylenediaminetetraacetic acid and salts, edetic acid

Coating Agents

sodium carboxymethylcellulose, cellulose acetate, cellulose acetatephthalate, ethylcellulose, gelatin, pharmaceutical glaze, hydroxypropylcellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulosephthalate, methacrylic acid copolymer, methylcellulose, polyethyleneglycol, polyvinyl acetate phthalate, shellac, sucrose, titanium dioxide,carnauba wax, microcystalline wax, zein

Colors

caramel, red, yellow, black or blends, ferric oxide

Complexing Agents

ethylenediaminetetraacetic acid and salts (EDTA), edetic acid, gentisicacid ethanolmaide, oxyquinoline sulfate

Desiccants

calcium chloride, calcium sulfate, silicon dioxide

Emulsifying and/or Solubilizing Agents

acacia, cholesterol, diethanolamine (adjunct), glyceryl monostearate,lanolin alcohols, lecithin, mono- and di-glycerides, monoethanolamine(adjunct), oleic acid (adjunct), oleyl alcohol (stabilizer), poloxamer,polyoxyethylene 50 stearate, polyoxyl 35 caster oil, polyoxyl 40hydrogenated castor oil, polyoxyl 10 oleyl ether, polyoxyl 20cetostearyl ether, polyoxyl 40 stearate, polysorbate 20, polysorbate 40,polysorbate 60, polysorbate 80, propylene glycol diacetate, propyleneglycol monostearate, sodium lauryl sulfate, sodium stearate, sorbitanmonolaurate, soritan monooleate, sorbitan monopalmitate, sorbitanmonostearate, stearic acid, trolamine, emulsifying wax

Filtering Aids

powdered cellulose, purified siliceous earth

Flavors and Perfumes

anethole, benzaldehyde, ethyl vanillin, menthol, methyl salicylate,monosodium glutamate, orange flower oil, peppermint, peppermint oil,peppermint spirit, rose oil, stronger rose water, thymol, tolu balsamtincture, vanilla, vanilla tincture, vanillin

Glidant and/or Anticaking Agents

calcium silicate, magnesium silicate, colloidal silicon dioxide, talc

Humectants

glycerin, hexylene glycol, propylene glycol, sorbitol

Ointment Bases

lanolin, anhydrous lanolin, hydrophilic ointment, white ointment, yellowointment, polyethylene glycol ointment, petrolatum, hydrophilicpetrolatum, white petrolatum, rose water ointment, squalane

Plasticizers

castor oil, diacetylated monoglycerides, diethyl phthalate, glycerin,mono- and di-acetylated monoglycerides, polyethylene glycol, propyleneglycol, triacetin, triethyl citrate

Polymer Membranes

cellulose acetate

Solvents

acetone, alcohol, diluted alcohol, amylene hydrate, benzyl benzoate,butyl alcohol, carbon tetrachloride, chloroform, corn oil, cottonseedoil, ethyl acetate, glycerin, hexylene glycol, isopropyl alcohol, methylalcohol, methylene chloride, methyl isobutyl ketone, mineral oil, peanutoil, polyethylene glycol, propylene carbonate, propylene glycol, sesameoil, water for injection, sterile water for injection, sterile water forirrigation, purified water

Sorbents

powdered cellulose, charcoal, purified siliceous earth

Carbon Dioxide Sorbents

barium hydroxide lime, soda lime

Stiffening Agents

hydrogenated castor oil, cetostearyl alcohol, cetyl alcohol, cetylesters wax, hard fat, paraffin, polyethylene excipient, stearyl alcohol,emulsifying wax, white wax, yellow wax

Suppository Bases

cocoa butter, hard fat, polyethylene glycol

Suspending and/or Viscosity-increasing Agents

acacia, agar, alginic acid, aluminum monostearate, bentonite, purifiedbentonite, magma bentonite, carbomer 934p, carboxymethylcellulosecalcium, carboxymethylcellulose sodium, carboxymethycellulose sodium 12,carrageenan, microcrystalline and carboxymethylcellulose sodiumcellulose, dextrin, gelatin, guar gum, hydroxyethyl cellulose,hydroxypropyl cellulose, hydroxypropyl methylcellulose, magnesiumaluminum silicate, methylcellulose, pectin, polyethylene oxide,polyvinyl alcohol, povidone, propylene glycol alginate, silicon dioxide,colloidal silicon dioxide, sodium alginate, tragacanth, xanthan gum

Sweetening Agents

aspartame, dextrates, dextrose, excipient dextrose, fructose, mannitol,saccharin, calcium saccharin, sodium saccharin, sorbitol, solutionsorbitol, sucrose, compressible sugar, confectioner's sugar, syrup

Tablet Binders

acacia, alginic acid, sodium carboxymethylcellulose, microcrystallinecellulose, dextrin, ethylcellulose, gelatin, liquid glucose, guar gum,hydroxypropyl methylcellulose, methycellulose, polyethylene oxide,povidone, pregelatinized starch, syrup

Tablet and/or Capsule Diluents

calcium carbonate, dibasic calcium phosphate, tribasic calciumphosphate, calcium sulfate, microcrystalline cellulose, powderedcellulose, dextrates, dextrin, dextrose excipient, fructose, kaolin,lactose, mannitol, sorbitol, starch, pregelatinized starch, sucrose,compressible sugar, confectioner's sugar

Table Disintegrants

alginic acid, microcrystalline cellulose, croscarmellose sodium,corspovidone, polacrilin potassium, sodium starch glycolate, starch,pregelatinized starch

Tablet and/or Capsule Lubricants

calcium stearate, glyceryl behenate, magnesium stearate, light mineraloil, polyethylene glycol, sodium stearyl fumarate, stearic acid,purified stearic acid, talc, hydrogenated vegetable oil, zinc stearate

Tonicity Agent

dextrose, glycerin, mannitol, potassium chloride, sodium chloride

Vehicle: Flavored and/or Sweetened

aromatic elixir, compound benzaldehyde elixir, iso-alcoholic elixir,peppermint water, sorbitol solution, syrup, tolu balsam syrup

Vehicle: oleaginous

almond oil, corn oil, cottonseed oil, ethyl oleate, isopropyl myristate,isopropyl palmitate, mineral oil, light mineral oil, myristyl alcohol,octyldodecanol, olive oil, peanut oil, persic oil, seame oil, soybeanoil, squalane

Vehicle: Solid Carrier

sugar spheres

Vehicle: Sterile

Bacteriostatic water for injection, bacteriostatic sodium chlorideinjection

Viscosity-increasing (see Suspending Agent)

Water Repelling Agent

cyclomethicone, dimethicone, simethicone

Wetting and/or Solubilizing Agent

benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride,docusate sodium, nonoxynol 9, nonoxynol 10, octoxynol 9, poloxamer,polyoxyl 35 castor oil, polyoxyl 40, hydrogenated castor oil, polyoxyl50 stearate, polyoxyl 10 oleyl ether, polyoxyl 20, cetostearyl ether,polyoxyl 40 stearate, polysorbate 20, polysorbate 40, polysorbate 60,polysorbate 80, sodium lauryl sulfate, sorbitan monolaureate, sorbitanmonooleate, sorbitan monopalmitate, sorbitan monostearate, tyloxapol

Preferred ingredients or excipients include: Salts of 1) amino acidssuch as glycine, arginine, aspartic acid, glutamic acid, lysine,asparagine, glutamine, proline, 2) carbohydrates, e.g. monosaccharidessuch as glucose, fructose, galactose, Mannose, arabinose, xylose, riboseand 3) disaccharides, such as lactose, trehalose, maltose, sucrose and4) polysaccharides, such as maltodextrins, dextrans, starch, glycogenand 5) alditols, such as mannitol, xylitol, lactitol, sorbitol 6)glucuronic acid, galacturonic acid, 7) cyclodextrins, such as methylcyclodextrin, hydroxypropyl-β-cyclodextrin and alike 8) inorganic salts,such as sodium chloride, potassium chloride, magnesium chloride,phosphates of sodium and potassium, boric acid ammonium carbonate andammonium phosphate, and 9) organic salts, such as acetates, citrate,ascorbate, lactate 10) emulsifying or solubilizing agents like acacia,diethanolamine, glyceryl monostearate, lecithin, monoethanolamine, oleicacid , oleyl alcohol, poloxamer, polysorbates, sodium lauryl sulfate,stearic acid, sorbitan monolaurate, sorbitan monostearate, and othersorbitan derivatives, polyoxyl derivatives, wax, polyoxyethylenederivatives, sorbitan derivatives 11) viscosity increasing reagentslike, agar, alginic acid and its salts, guar gum, pectin, polyvinylalcohol, polyethylene oxide, cellulose and its derivatives propylenecarbonate, polyethylene glycol, hexylene glycol, tyloxapol. A furtherpreferred group of excipients or ingredients includes sucrose,trehalose, lactose, sorbitol, lactitol, inositol, salts of sodium andpotssium such as acetate, phosphates, citrates, borate, glycine,arginine, polyethylene oxide, polyvinyl alcohol, polyethylene glycol,hexylene glycol, methoxy polyethylene glycol, gelatin,hydroxypropyl-β-cyclodextrin.

Polymer—a large molecule built up by the repetition of small, simplechemical units. The repeating units may be linear or branched to forminterconnected networks. The repeat unit is usually equivalent or nearlyequivalent to the monomer.

Polymeric carriers—polymers used for encapsulation of protein crystalsfor delivery of proteins, including biological delivery. Such polymersinclude biocompatible and biodegradable polymers. The polymeric carriermay be a single polymer type or it may be composed of a mixture ofpolymer types. Polymers useful as the polymeric carrier, include forexample, poly (acrylic acid), poly (cyanoacrylates), poly (amino acids),poly (anhydrides), poly (depsipeptide), poly (esters) such as poly(lactic acid) or PLA, poly (lactic-co-glycolic acid) or PLGA, poly(B-hydroxybutryate), poly (caprolactone) and poly (dioxanone); poly(ethylene glycol), poly ((hydroxypropyl)methacrylamide, poly[(organo)phosphazene], poly (ortho esters), poly (vinyl alcohol), poly(vinylpyrrolidone), maleic anhydride-alkyl vinyl ether copolymers,pluronic polyols, albumin, natural and synthetic polypeptides, alginate,cellulose and cellulose derivatives, collagen, fibrin, gelatin,hyaluronic acid, oligosaccharides, glycaminoglycans, sulfatedpolysaccharides, or any conventional material that will encapsulateprotein crystals.

Protein—a complex high polymer containing carbon, hydrogen, oxygen,nitrogen and usually sulfur and composed of chains of amino acidsconnected by peptide linkages. Proteins in this application refer toglycoproteins, antibodies, non-enzyme proteins, enzymes, hormones andpeptides. The molecular weight range for proteins includes peptides of1000 Daltons to glycoproteins of 600 to 1000 kiloDaltons. Smallproteins, less than 10,000 Daltons, may be too small to be characterizedby a highly organized tertiary structure, wherein said tertiarystructure is organized around a hydrophobic core.

In one embodiment of this invention, such proteins have a molecularweight of greater than or equal to 10,000 Daltons. According to analternate embodiment, that molecular weight is greater than or equal to20,000 Daltons. According to another alternate embodiment, thatmolecular weight is greater than or equal to 30,000 Daltons. Accordingto a further alternate embodiment, that molecular weight is greater thanor equal to 40,000 Daltons. According to another alternate embodiment,that molecular weight is greater than or equal to 50,000 Daltons.

Protein activity—an activity selected from the group consisting ofbinding, catalysis, signaling, transport, or other activities whichinduce a functional response within the environment in which the proteinis used, such as induction of immune response, enzymatic activity, orcombinations thereof.

Protein activity release rate—the quantity of protein dissolved per unittime.

Protein crystal—protein molecules arranged in a crystal lattice. Proteincrystals contain a pattern of specific protein—protein interactions thatare repeated periodically in three dimensions. The protein crystals ofthis invention do not include amorphous solid forms or precipitates ofproteins, such as those obtained by lyophilizing a protein solution.

Protein crystal formulation—a combination of protein crystalsencapsulated within a polymeric carrier to form coated particles. Thecoated particles of the protein crystal formulation may have a sphericalmorphology and be microspheres of up to 500 micro meters in diameter orthey may have some other morphology and be microparticulates. For thepurposes of this application, “protein crystal formulations” areincluded in the term “compositions”.

Protein delivery system—one or more of a protein crystal formulation orcomposition, a process for making the formulation or a method ofadministering the formulation to biological entities or means therefor.

Protein loading—the protein content of microspheres, as calculated as apercentage by weight of protein relative to the weight of the dryformulation. A typical range of protein loading is from 1-80%.

Protein release—the release of active protein from a polymeric carrier,as controlled by one or more of the following factors: (1) degradationof the polymer matrix; (2) rate of crystal dissolution within thepolymer matrix; (3) diffusion of dissolved protein through the polymermatrix; (4) protein loading; and (5) diffusion of biological medium intothe protein crystal/polymer matrix.

Prophylactically effective amount—an amount of a protein or nucleic acidcrystal or crystal formulation or composition which is effective toprevent a condition in a living organism to whom it is administered oversome period of time.

Reconstitution—dissolution of protein or nucleic acid crystals orcrystal formulations or compositions in an appropriate buffer orpharmaceutical formulation.

Shelf stability—the loss of specific activity and/or changes insecondary structure from the native protein over time incubated underspecified conditions.

Stability—the loss of specific activity and/or changes in secondarystructure from the native protein over time while in solution underspecified conditions.

Stabilization—the process of preventing the loss of specific activityand/or changes in secondary structure from the native proteins, bypreparing formulations of protein crystals or DNA crystals or RNAcrystals with excipients or ingredients.

Therapeutic protein—a protein as described above, which is administeredto a living organism in a formulation or composition or a pharmaceuticalformulation or composition. Therapeutic proteins include all of theprotein types described herein.

Vaccine antigen—a protein derived from a pathogenic agent such as avirus, parasite, bacteria or tumor cell. The protein activity of suchvaccine antigens is the induction of protective immune responsesspecific for a pathogenic agent or tumor.

Crystallinity

Crystallinity of macromolecules is of great value for their storage anddelivery in vivo. However, few techniques exist for the preparation oflarge quantities of such crystalline macromolecules which are stableoutside of the mother liquor. Crystals of proteins and nucleic acidsmust be handled with considerable care, since they are extremely fragileand contain a high proportion of solvent. It is well known in x-raycrystallography that the diffraction patterns from macromolecularcrystals quickly degenerate upon dehydration in air. Normally, a crystalis carefully separated from its mother liquor and inserted into acapillary tube. The tube is sealed from the air using dental wax orsilicone grease, along with a small amount of mother liquor inside tomaintain hydration [McPherson, A., Preparation and Analysis of ProteinCrystals, Robert E. Krieger Publishing, Malabar, p. 214 (1989)]. Anothertechnique is to collect data from macromolecular crystals at cryogenictemperatures. The crystals are prepared and then rapidly cooled toprevent ice lattice formation in the aqueous medium. Instead of ice, arigid glass forms, encasing the crystal with little damage. Crystals arethen maintained at 100° K to prevent crystal disintegrations [Rodgers,D. W., in Methods in Enzymology (Eds., Carter, C. W. and Sweet, R. M.)Academic Press, v.276, p. 183 (1997)]. While this technique allows oneto maintain crystals outside of their mother liquor, it cannot be usedat temperatures higher than 100° K.

In principle, dried crystals can be prepared by lyophilization. However,this technique involves rapid cooling of the material and can be appliedonly to freeze stable products. The aqueous solution is first frozen tobetween −40 and −50° C. Then, the ice is removed under vacuum. Iceformation is usually destructive to the protein crystal lattice,yielding a mixture of crystals and amorphous precipitate.

It is desirable to produce macromolecules, in the crystalline state,that are pure and stable under storage conditions at ambienttemperatures. Such crystals constitute a particularly advantageous formof proteins or nucleic acids for dosage preparations of therapeutics andvaccines. The present invention provides formulations and compositionsfor storage of crystalline macromolecules as either solid particles ordispersed in a non-aqueous solvent. Furthermore, the invention may beapplied to the storage of a single biological macromolecule or a mixtureof macromolecules that do not interact with each other.

In another embodiment, this invention provides a method for renderingbiological macromolecules suitable for storage in suspensions comprisingreplacing the mother liquor with a non-aqueous solvent. In yet anotherembodiment, the crystalline slurry can be rendered solid by spinning outthe first solvent and washing the remaining crystalline solid using asecond organic solvent to remove water, followed by evaporation of thenon-aqueous solvent.

Non-aqueous slurries of crystalline therapeutic proteins are especiallyuseful for subcutaneous delivery, while solid formulations are ideallysuited for pulmonary administration. Pulmonary delivery is particularlyuseful for biological macromolecules which are difficult to deliver byother routes of administration. (See, for example, PCT patentapplications Wo 96/32152, WO 95/24183 and WO 97/41833).

The proteins referred to below include protein crystals themselves, ornucleic acid crystals comprising DNA or RNA which encode those proteinsupon cellular uptake.

This invention advantageously provides compositions and formulations ofcrystals of proteins or nucleic acids.

Stability of Encapsulated Crystals

Those of skill in the art will appreciate that protein stability is oneof the most important obstacles to successful formulation of polymermicroparticulate delivery systems that control the release of proteins.The stability of proteins encapsulated in polymeric carriers may bechallenged at three separate stages: manufacture of the protein crystalcomposition, protein release from the resulting composition and in vivostability after the protein release. During preparation ofmicroparticles or microspheres containing soluble or amorphous proteins,the use of organic solvents and lyophilization are especiallydetrimental to protein stability. Subsequently, released proteins aresusceptible to moisture-induced aggregation, thus resulting in permanentinactivation.

In order to achieve high protein stability during preparation of proteinformulations and compositions according to the present invention, it isnecessary to restrict the mobility of individual protein molecules—aresult best achieved in the crystalline solid state. For the purpose ofthis application, solid state may be divided into two categories:amorphous and crystalline. The three-dimensional long-range order thatnormally exists in a crystalline material does not exist in theamorphous state. Furthermore, the position of molecules relative to oneanother is more random in the amorphous or liquid states, relative tothe highly ordered crystalline state. Thus, amorphous proteins may beless stable than their crystalline counterparts.

FIG. 1 depicts the relative stability of the following molecular statesof Candida rugosa lipase (“CRL”): crosslinked amorphous, liquid,crystalline in 20% organic solvent, crosslinked crystalline in 20%organic solvent, crosslinked crystalline without organic solvent. FIG. 1shows that crystalline CRL retains 80% activity for more than 175 hoursin 20% organic solvent. In contrast, both amorphous and soluble forms ofthe enzyme are completely inactivated within hours. The presentinvention advantageously utilizes the crystalline forms of proteinsbecause of their superior stability characteristics.

Maintaining Crystallinity

In order to use protein crystals as the protein source for preparingprotein formulations and compositions according to the presentinvention, the problem of protein crystal dissolution outside thecrystallization solution (“mother liquor”) had to be overcome. In orderto maintain protein crystallinity and hence stability, in the productionof the protein crystal formulations and compositions of this invention,several approaches may be used:

1. Crystals remain in the mother liquor in the course of producingprotein crystals encapsulated with polymeric carriers. Many compoundsused in protein crystallization, such as salts, PEG and organicsolvents, are compatible with polymer processing conditions.

2. Kinetics of dissolution. The rate of crystal dissolution outside themother liquor depends on conditions, such as pH, temperature, presenceof metal ions, such as Zn, Cu and Ca and concentration of precipitants.By varying these conditions, one can slow down the dissolution ofcrystals for several hours. At the same time, the process ofmicroparticulate formation is very fast and normally takes seconds tominutes to complete.

3. Dried protein crystals. The mother liquor can be removed byfiltration and the remaining crystalline paste can be dried by air,under vacuum, by washing with water miscible organic solvents and/or bylyophilization.

4. Protein crystals can be chemically crosslinked to formnon-dissolvable or slowly dissolvable crystals.

5. The crystal size and shape can be manipulated and controlled in thecourse of crystallization.

Thus, a range of crystal morphologies, each having different dissolutionkinetics and subsequently different sustained release profiles comparedto amorphous proteins, is available.

Protein Constituents

The protein constituents of the formulations and compositions of thisinvention may be those which are naturally or synthetically modified.They may be glycoproteins, phosphoproteins, sulphoproteins,iodoproteins, methylated proteins, unmodified proteins or contain othermodifications. Such protein constituents may be any protein, including,for example, therapeutic proteins, prophylactic proteins, includingantibodies, cleaning agent proteins, including detergent proteins,personal care proteins, including cosmetic proteins, veterinaryproteins, food proteins, feed proteins, diagnostic proteins anddecontamination proteins.

In one embodiment of this invention, such proteins have a molecularweight of greater than or equal to 10,000 Daltons. According to analternate embodiment, that molecular weight is greater than or equal to20,000 Daltons. According to another alternate embodiment, thatmolecular weight is greater than or equal to 30,000 Daltons. Accordingto a further alternate embodiment, that molecular weight is greater thanor equal to 40,000 Daltons. According to another alternate embodiment,that molecular weight is greater than or equal to 50,000 Daltons.

Included among such proteins are enzymes, such as, for example,hydrolases, isomerases, lyases, ligases, adenylate cyclases,transferases and oxidoreductases. Examples of hydrolases includeelastase, esterase, lipase, nitrilase, amylase, pectinase, hydantoinase,asparaginase, urease, subtilisin, thermolysin and other proteases andlysozyme. Examples of lyases include aldolases and hydroxynitrile lyase.Examples of oxidoreductases include peroxidase, laccase, glucoseoxidase, alcohol dehydrogenase and other dehydrogenases. Other enzymesinclude cellulases and oxidases.

Examples of therapeutic or prophylactic proteins include hormones suchas insulin, glucogon-like peptide 1 and parathyroid hormone, antibodies,inhibitors, growth factors, postridical hormones, nerve growth hormones,blood clotting factors, adhesion molecules, bone morphogenic proteinsand lectins trophic factors, cytokines such as TGF-β, IL-2, IL-4, α-IFN,β-IFN, γ-IFN, TNF, IL-6, IL-8, lymphotoxin, IL-5, Migration inhibitionfactor, GMCSF, IL-7, IL-3, monocyte-macrophage colony stimulatingfactors, granulocyte colony stimulating factors, multidrug resistanceproteins, other lymphokines, toxoids, erythropoietin, Factor VIII,amylin, TPA, dornase-α,α-1-antitripsin, human growth hormones, nervegrowth hormones, bone morphogenic proteins, urease, toxoids, fertilityhormones, FSH and LSH.

Therapeutic proteins, such as the following, are also included:

leukocyte markers, such as CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD11a,CD11b, CD11c, CD13, CD14, CD18, CD19, CE20, CD22, CD23, CD27 and itsligand, CD28 and its ligands B7.1, B7.2, B7.3, CD29 and its ligands,CD30 and its ligand, CD40 and its ligand gp39, CD44, CD45 and isoforms,Cdw52 (Campath antigen), CD56, CD58, CD69, CD72, CTLA-4, LFA-1 and TCR

histocompatibility antigens, such as MHC class I or II antigens, theLewis Y antigens, SLex, SLey, SLea and SLeb;

integrins, such as VLA-1, VLA-2, VLA-3, VLA-4, VLA-5, VLA-6 and LFA-1;

adhesion molecules, such as Mac-1 and p150,95;

selectins, such as L-selectin, P-selectin and E-selectin and theircounterreceptors VCAM-1, ICAM-1, ICAM-2 and LFA-3;

interleukins, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,IL-10, IL-11, IL-12, IL-13, IL-14 and IL-15;

interleukin receptors, such as IL-1R, IL-2R, IL-4R, IL-5R, IL-6R, IL-7R,IL-8R, IL-10R, IL-11R, IL-12R, IL-13R, IL-14R and IL-15R;

chemokines, such as PF4, RANTES, MIP1α, MCP1, NAP-2, Groα, Groβ andIL-8;

growth factors, such as TNFalpha, TGFbeta, TSH, VEGF/VPF, PTHrP, EGFfamily, EGF, PDGF family, endothelin and gastrin releasing peptide(GRP);

growth factor receptors, such as TNFalphaR, RGFbetaR, TSHR, VEGFR/VPFR,FGFR, EGFR, PTHrPR, PDGFR family, EPO-R; GCSF-R and other hematopoieticreceptors;

interferon receptors, such as IFNαR, IFNβR and IFNγR;

Igs and their receptors, such as IgE, FceRI and FceRII;

blood factors, such as complement C3b, complement C5a, complement C5b-9,Rh factor, fibrinogen, fibrin and myelin associated growth inhibitor.

The protein constituent of the formulations and compositions of thisinvention may be any natural, synthetic or recombinant protein antigenincluding, for example, tetanus toxoid, diptheria toxoid, viral surfaceproteins, such as CMV glycoproteins B, H and gCIII, HIV-1 envelopeglycoproteins, RSV envelope glycoproteins, HSV envelope glycoproteins,EBV envelope glycoproteins, VZV envelope glycoproteins, HPV envelopeglycoproteins, Influenza virus glycoproteins, Hepatitis family surfaceantigens; viral structural proteins, viral enzymes, parasite proteins,parasite glycoproteins, parasite enzymes and bacterial proteins.

Also included are tumor antigens, such as her2-neu, mucin, CEA andendosialin. Allergens, such as house dust mite antigen, lol p1 (grass)antigens and urushiol are included.

Toxins, such as pseudomonas endotoxin and osteopontin/uropontin, snakevenom and bee venom are included.

Also included are glycoprotein tumor-associated antigens, for example,carcinoembryonic antigen (CEA), human mucins, her-2/neu andprostate-specific antigen (PSA) [R. A. Henderson and O. J. Finn,Advances in Immunology, 62, pp. 217-56 (1996)].

Administration and Biological Delivery

To date, therapeutic proteins have generally been administered byfrequent injection, due to their characteristic negligible oralbioavailability and short plasma life. The protein crystal formulationsand compositions of the present invention, which includemicroparticulate-based sustained release systems for protein drugs,advantageously permit improved patient compliance and convenience, morestable blood levels and potential dose reduction. The slow and constantrelease capabilities of the present invention advantageously permitreduced dosages, due to more efficient delivery of active protein.Significant cost savings may be achieved by using the proteinformulations and compositions described herein.

Formulations and compositions comprising protein crystals in polymericdelivery carriers according to this invention may also comprise anyconventional carrier or adjuvant used in vaccines, pharmaceuticals,personal care formulations and compositions, veterinary formulations, ororal enzyme supplementation. These carriers and adjuvants include, forexample, Freund's adjuvant, ion exchangers, alumina, aluminum stearate,lecithin, buffer substances, such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts or electrolytes, such as protamine sulfate,disodium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium, trisilicate, cellulose-based substances andpolyethylene glycol. Adjuvants for topical or gel base forms mayinclude, for example, sodium carboxymethylcellulose, polyacrylates,polyoxyethylene-polyoxypropylene-block polymers, polyethylene glycol andwood wax alcohols.

According to one embodiment of this invention, protein crystals may becombined with any conventional materials used for controlled releaseadministration, including pharmaceutical controlled releaseadministration. Such materials include, for example, coatings, shellsand films, such as enteric coatings and polymer coatings and films.

Protein formulations in polymeric delivery carriers and compositions(compositions) according to this invention, which may be devices, suchas implantable devices and may be microparticulate protein deliverysystems.

In one embodiment of this invention, the macromolecule crystals have alongest dimension between about 0.01 μm and about 500 μm, alternativelybetween about 0.1 μm and about 100 μm. The most preferred embodiment isthat the protein crystal of protein crystal formulation components arebetween about 50 μm and about 100 μm in their longest dimension. Suchcrystals may have a shape selected from the group consisting of:spheres, needles, rods, plates, such as hexagons and squares, rhomboids,cubes, bipyramids and prisms.

According to the present invention, encapsulation of protein crystals orprotein crystal formulations in polymeric carriers to make compositionsmay be carried out on protein crystals which are crosslinked oruncrosslinked. Such protein crystals may be obtained commercially orproduced as illustrated herein.

Protein or nucleic acid crystals or crystal formulations andcompositions according to this invention may be used as ingredients inpersonal care compositions, including cosmetics, such as creams,lotions, emulsions, foams, washes, compacts, gels, mousses, slurries,powders, sprays, pastes, ointments, salves, balms, drops, shampoos andsunscreens. In topical creams and lotions, for example, they may be usedas humectants or for skin protection, softening, bleaching, cleaning,deproteinization, lipid removal, moisturizing, decoloration, colorationor detoxification. They may also be used as anti-oxidants in cosmetics.

According to this invention, any individual, including humans, animalsand plants, may be treated in a pharmaceutically acceptable manner witha pharmaceutically effective amount of protein or nucleic acid crystalsor a crystal formulation or composition for a period of time sufficientto treat a condition in the individual to whom they are administeredover some period of time. Alternatively, individuals may receive aprophylactically effective amount of protein or nucleic acid crystals orcrystal formulation or composition of this invention which is effectiveto prevent a condition in the individual to whom they are administeredover some period of time.

Protein or nucleic acid crystals or crystal formulations or compositionsmay be administered alone, as part of a pharmaceutical, personal care orveterinary preparation, or as part of a prophylactic preparation, suchas a vaccine, with or without adjuvant. They may be administered byparenteral or oral routes. For example, they may be administered byoral, pulmonary, nasal, aural, anal, dermal, ocular, intravenous,intramuscular, intraarterial, intraperitoneal, mucosal, sublingual,subcutaneous, or intracranial route. In either pharmaceutical, personalcare or veterinary applications, protein or nucleic acid crystal orcrystal formulations or compositions may be topically administered toany epithelial surface. Such epithelial surfaces include oral, ocular,aural, anal and nasal surfaces, which may be treated, protected,repaired or detoxified by application of protein or nucleic acidcrystals or crystal formulations or compositions.

Pharmaceutical, personal care, veterinary or prophylactic formulationsand compositions comprising protein or nucleic acid crystal or crystalformulations or compositions according to this invention may also beselected from the group consisting of tablets, liposomes, granules,spheres, microparticles, microspheres and capsules.

For such uses, as well as other uses according to this invention,protein or nucleic acid crystals or crystal formulations andcompositions may be formulated into tablets. Such tablets constitute aliquid-free, dust-free form for storage of protein or nucleic acidcrystal or crystal formulations or compositions which are easily handledand retain acceptable levels of activity or potency.

Alternatively, protein or nucleic acid crystals or crystal formulationsor compositions may be in a variety of conventional depot forms employedfor administration to provide reactive compositions. These include, forexample, solid, semi-solid and liquid dosage forms, such as liquidsolutions or suspensions, slurries, gels, creams, balms, emulsions,lotions, powders, sprays, foams, pastes, ointments, salves, balms anddrops.

Protein or nucleic acid crystals on formulations or compositionsaccording to this invention may also comprise any conventional carrieror adjuvant used in pharmaceuticals, personal care compositions orveterinary formulations. These carriers and adjuvants include, forexample, Freund's adjuvant, ion exchangers, alumina, aluminum stearate,lecithin, buffer substances, such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts or electrolytes, such as protamine sulfate,disodium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium, trisilicate, cellulose-based substances andpolyethylene glycol. Adjuvants for topical or gel base forms mayinclude, for example, sodium carboxymethylcellulose, polyacrylates,polyoxyethylene-polyoxypropylene-block polymers, polyethylene glycol andwood wax alcohols.

The most effective mode of administration and dosage regimen of theprotein or nucleic acid crystals or crystal formulations or compositionsof this invention will depend on the effect desired, previous therapy,if any, the individual's health status or status of the condition itselfand response to the protein or nucleic acid crystals or crystalformulations or compositions and the judgment of the treating physicianor clinician. The protein or nucleic acid crystals or crystalformulations or compositions may be administered in any dosage formacceptable for pharmaceuticals, vaccinations, gene therapy,immunotherapy, personal care compositions or veterinary formulations, atone time or over a series of treatments.

The amount of the protein or nucleic acid crystals or crystalformulations or compositions which provides a single dosage will varydepending upon the particular mode of administration, formulation, doselevel or dose frequency. A typical preparation will contain betweenabout 0.01% and about 99%, preferably between about 1% and about 50%,protein or nucleic acid crystals (w/w). Alternatively, a preparationwill contain between about 0.01% and about 80% protein crystals,preferably between about 1% and about 50%, protein crystals (w/w).Alternatively, a preparation will contain between about 0.01% and about80% protein crystal formulation, preferably between about 1% and about50%, protein crystal formulation(w/w).

Upon improvement of the individual's condition, a maintenance dose ofprotein or nucleic acid crystals or crystal formulations or compositionsmay be administered, if necessary. Subsequently, the dosage or frequencyof administration, or both, may be reduced as a function of thesymptoms, to a level at which the improved condition is retained. Whenthe condition has been alleviated to the desired level, treatment shouldcease. Individuals may, however, require intermittent treatment on along-term basis upon any recurrence of the condition or symptomsthereof.

Production of Crystals, Crystal Formulations and Compositions:

According to the one embodiment of this invention, crystals, crystalformulations and compositions are prepared by the following process:first, the protein or nucleic acid is crystallized. Next, excipients oringredients selected from sugars, sugar alcohols, viscosity increasingagents, wetting or solubilizing agents, buffer salts, emulsifyingagents, antimicrobial agents, antioxidants, and coating agents are addeddirectly to the mother liquor. Alternatively, the crystals are suspendedin an excipient solution, after the mother liquor is removed, for aminimum of 1 hour to a maximum of 24 hours. The excipient concentrationis typically between about 0.01 to 30% W/W, which corresponds to acrystal concentration of 99.99 to 70% W/W, respectively. Mostpreferably, the excipient concentration is between about 0.1 to 10%,which corresponds to a crystal concentration of 99.9 to 90% W/W,respectively. The ingredient concentration is between about 0.01 to 90%.The crystal concentration is between about 0.01 to 95%. The motherliquor is then removed from the crystal slurry either by filtration orby centrifugation. Subsequently, the crystals are washed optionally withsolutions of 50 to 100% one or more organic solvents such as, forexample, ethanol, methanol, isopropanol or ethyl acetate, either at roomtemperature or at temperatures between −20°C to 25°C. The crystals arethe dried either by passing a stream of nitrogen, air, or inert gas overthe crystals. Alternatively, the crystals are dried by air drying or bylyophilization or by vacuum drying. The drying is carried out for aminimum 1 hour to a maximum of 72 hours after washing, until themoisture content of the final product is below 10% by weight, mostpreferably below 5%. Finally, micronizing of the crystals can beperformed if necessary.

According to one embodiment of this invention, when preparing proteincrystals, protein crystal formulations or compositions, enhancers, suchas surfactants are not added during crystallization. Excipients oringredients are added to the mother liquor after crystallization, at aconcentration of between about 1-10% W/W, alternatively at aconcentration of between about 0.1-25% W/W, alternatively at aconcentration of between about 0.1-50% W/W. These concentrationscorrespond to crystal concentrations of 99-90% W/W, 99.9-75% W/W and99.9-50% W/W, respectively. The excipient or ingredient is incubatedwith the crystals in the mother liquor for about 0.1-3 hrs,alternatively the incubation is carried out for 0.1-12 hrs,alternatively the incubation is carried out for 0.1-24 hrs.

In another embodiment of this invention, the ingredient or excipient isdissolved in a solution other than the mother liquor, and the proteincrystals are removed from the mother liquor and suspended in theexcipient or ingredient solution. The ingredient or excipientconcentrations and the incubation times are the same as those describedabove.

Slow Release Forms and Vaccines

In another embodiment of this invention, encapsulation of lipases inpolymeric carriers provide compositions useful to treat patientssuffering from intestinal lipase deficiency. Such patients include thosewith pancreatic steatorrhea, due to advanced pancreatic insufficiencyrequire oral lipase supplementation. Unfortunately, current therapeuticmethods may not be flexible enough to protect the active lipase duringtransit through the gastro-intestinal tract and to release the enzymeactivity where it is critically needed in the small bowel (See L.Guarner et al., “Fate of oral enzymes in pancreatic insufficiency,” Gut,vol. 34, pp. 708-712, (1993)). The flexibility of the present inventionin preparing slowly available active lipase solves the present problemsoften associated with lipase supplementation. According to oneembodiment of this invention, the combination of encapsulated lipasecrystals (compositions) and unencapsulated crosslinked lipase crystalsor formulations provides a drug therapy regime in which enzyme activityis available early on from the unencapsulated crosslinked lipase. Asthis material undergoes proteolytic degradation, the encapsulated enzyme(composition) begins to release enzyme activity into the more distalbowel. A similar strategy may be used to solve other enzyme ortherapeutic protein supplementation problems.

The present invention may also utilize other slow release methodologies,such as silicon based rings or rods which have been preloaded withencapsulated protein crystals containing hormones, antibodies or enzymesor compositions containing them. The purpose of this technique is toprovide a constant level of protein to the bloodstream over a period ofweeks or months. Such implants can be inserted intradermally and can besafely replaced and removed when needed.

Other formulations and compositions according to this invention includevaccine formulations and compositions comprising protein (antigen)crystals, adjuvant and encapsulating polymer(s). The protein antigen maybe a viral glycoprotein, viral structural protein, viral enzyme,bacterial protein, or some engineered homolog of a viral or bacterialprotein, or any immunopotentiating protein, such as a cytokine. Oneembodiment of such formulations or compositions involves a singlevaccine injection containing microspheres having three or more differentrelease profiles. In this way, antigen formulations or composition maybe released over a sustained period sufficient to generate lastingimmunity. By virtue of this formulation or composition, multiple antigenboosts may be in single unit form. The faster degrading preparation(composition) may contain an immunogenic adjuvant to enhance the immuneresponse. One advantage of such a system is that by using proteincrystals, the native three-dimensional structures of the epitopes aremaintained and presented to the immune system in their native form.

Once the immune system is primed, there may be less need for an adjuvanteffect. Therefore, in the slower degrading inoculations, a lessimmunogenic adjuvant may be included and possibly no adjuvant may berequired in the slowest degrading microspheres of the formulations andcompositions . In this way, patient populations in remote areas will nothave to be treated multiple times in order to provide protection againstinfectious diseases. One of skill in the art of biological delivery ofproteins will appreciate that many variations on this theme arefeasible. Accordingly, the examples provided here are not intended tolimit the invention.

In another embodiment of this invention, a combination vaccine could beproduced, whereby immunity to multiple diseases is induced in a singleinjection. As discussed above, microspheres having different releaseprofiles may be combined alone or in formulations and compositions andmay include microspheres containing antigens from multiple infectiousagents to produce a combination vaccine (formulations and compositions).For example, microspheres having multiple release profiles andcontaining antigen crystals of measles, mumps, rubella, polio andhepatitis B agents could be combined and administered to children.Alternatively, microspheres having multiple release profiles andcontaining crystals of different isolates of HIV gp120 could be combinedto produce a vaccine for HIV-1 or HIV-2.

Another advantage of the present invention is that the protein crystalsencapsulated within polymeric carriers and forming a compositioncomprising microspheres can be dried by lyophilization. Lyophilization,or freeze-drying allows water to be separated from the composition. Theprotein crystal composition is first frozen and then placed in a highvacuum. In a vacuum, the crystalline H₂O sublimes, leaving the proteincrystal composition behind containing only the tightly bound water. Suchprocessing further stabilizes the composition and allows for easierstorage and transportation at typically encountered ambienttemperatures.

This feature is especially desirable for therapeutic proteins andvaccines which can be dispensed into single dose sterile containers(“ampules”) or alternatively, any desired increment of a single dose asa slurry, in a formulation or a composition. The ampules containing thedispensed slurries, formulations or compositions can then be capped,batch frozen and lyophilized under sterile conditions. Such sterilecontainers can be transported throughout the world and stored at ambienttemperatures. Such a system would be useful for providing sterilevaccines and therapeutic proteins to remote and undeveloped parts of theworld. At the point of use, the ampule is rehydrated with the sterilesolvent or buffer of choice and dispensed. Under this scenario, minimalor no refrigeration is required.

Protein Crystallization

Protein crystals are grown by controlled crystallization of protein fromaqueous solutions or aqueous solutions containing organic solvents.Solution conditions that may be controlled include, for example, therate of evaporation of solvent, organic solvents, the presence ofappropriate co-solutes and buffers, pH and temperature. A comprehensivereview of the various factors affecting the crystallization of proteinshas been published by McPherson, Methods Enzymol., 114, pp. 112-20(1985).

McPherson and Gilliland, J. Crystal Growth, 90, pp. 51-59 (1988) havecompiled comprehensive lists of proteins and nucleic acids that havebeen crystallized, as well as the conditions under which they werecrystallized. A compendium of crystals and crystallization recipes, aswell as a repository of coordinates of solved protein and nucleic acidstructures, is maintained by the Protein Data Bank at the BrookhavenNational Laboratory [http//www. pdb.bnl.gov; Bernstein et al., J. Mol.Biol., 112, pp. 535-42 (1977)]. These references can be used todetermine the conditions necessary for crystallization of a protein, asa prelude to the formation of appropriate protein crystals and can guidethe crystallization strategy for other proteins. Alternatively, anintelligent trial and error search strategy can, in most instances,produce suitable crystallization conditions for many proteins, providedthat an acceptable level of purity can be achieved for them [see, e.g.,C. W. Carter, Jr. and C. W. Carter, J. Biol. Chem., 254, pp. 12219-23(1979)].

In general, crystals are produced by combining the protein to becrystallized with an appropriate aqueous solvent or aqueous solventcontaining appropriate crystallization agents, such as salts or organicsolvents. The solvent is combined with the protein and may be subjectedto agitation at a temperature determined experimentally to beappropriate for the induction of crystallization and acceptable for themaintenance of protein activity and stability. The solvent canoptionally include co-solutes, such as divalent cations, co-factors orchaotropes, as well as buffer species to control pH. The need forco-solutes and their concentrations are determined experimentally tofacilitate crystallization.

It is critical to differentiate between amorphous precipitates andcrystalline material. Crystalline material is a form of the solid stateof matter, which is distinct from the amorphous solid state. Crystalsdisplay characteristic features including a lattice structure,characteristic shapes and optical properties such as refractive indexand birefringence. A crystal consists of atoms arranged in a patternthat repeats periodically in three dimensions. In contrast, amorphousmaterial is a non-crystalline solid form of matter, sometimes referredto as an amorphous precipitate. Such precipitates have no molecularlattice structure characteristic of the crystalline solid state and donot display birefringence or other spectroscopic characteristics typicalof the crystalline forms of matter.

In an industrial-scale process, the controlled precipitation leading tocrystallization can best be carried out by the simple combination ofprotein, precipitant, co-solutes and, optionally, buffers in a batchprocess. As another option, proteins may be crystallized by usingprotein precipitates as the starting material. In this case, proteinprecipitates are added to a crystallization solution and incubated untilcrystals form. Alternative laboratory crystallization methods, such asdialysis or vapor diffusion, can also be adopted. McPherson, supra andGilliland, supra, include a comprehensive list of suitable conditions intheir reviews of the crystallization literature.

Occasionally, in cases in which the crystallized protein is to becrosslinked, incompatibility between an intended crosslinking agent andthe crystallization medium might require exchanging the crystals into amore suitable solvent system.

Many of the proteins for which crystallization conditions have alreadybeen described, may be used to prepare protein crystals according tothis invention. It should be noted, however, that the conditionsreported in most of the above-cited references have been optimized toyield, in most instances, a few large, diffraction quality crystals.Accordingly, it will be appreciated by those of skill in the art thatsome degree of adjustment of these conditions to provide a high yieldingprocess for the large scale production of the smaller crystals used inmaking protein crystals the present invention may be necessary.

Crosslinking of Protein Crystals

According to one embodiment of this invention, for example, the releaserate of the protein from the polymeric carrier (composition) may beslowed and controlled by using protein crystals that have beenchemically crosslinked using a crosslinker, such as for example, abiocompatible crosslinker. Thus, once protein crystals have been grownin a suitable medium they may be crosslinked.

Crosslinking may be carried out using reversible crosslinkers, inparallel or in sequence. The resulting crosslinked protein crystals arecharacterized by a reactive multi-functional linker, into which atrigger is incorporated as a separate group. The reactive functionalityis involved in linking together reactive amino acid side chains in aprotein and the trigger consists of a bond that can be broken byaltering one or more conditions in the surrounding environment (e.g.,pH, temperature, or thermodynamic water activity). This is illustrateddiagrammatically as:

X−Y−Z+2 AA residues—>AA₁−X−Y−Z−AA₂

change in environment—>AA₁−X+Y−Z−AA₂

where X and Z are groups with reactive functionality

where Y is a trigger

where AA₁ and AA₂ represent reactive amino acid residues on the sameprotein or on two different proteins. The bond between the crosslinkingagent and the protein may be a covalent or ionic bond, or a hydrogenbond. The change in surrounding environment results in breaking of thetrigger bond and dissolution of the protein. Thus, when the crosslinkswithin protein crystals crosslinked with such reversible crosslinkingagents break, dissolution of protein crystal begins and therefore therelease of activity.

Alternatively, the reactive functionality of the crosslinker and thetrigger may be the same, as in:

X−Z+2AA residues—>AA₁−X−Z−AA₂

change in environment —>AA₁+X−Z−AA₂.

The crosslinker may be homofunctional (X=Y) or heterofunctional (X isnot equal to Y). The reactive functionality X and Y may be, but notlimited to the following functional groups (where R, R′, R″ and R′″ maybe alkyl, aryl or hydrogen groups):

I. Reactive acyl donors are exemplified by: carboxylate esters RCOOR′,amides RCONHR′, Acyl azides RCON₃, carbodiimides R−N═C=N−R′,N-hydroxyimide esters, RCO—O—NR′, imidoesters R—C═NH2⁺(OR′), anhydridesRCO—O—COR′, carbonates RO—CO—O—R′, urethanes RNHCONHR′, acid halidesRCOHal (where Hal=a halogen), acyl hydrazides RCONNR′R″, O-acylisoureasRCO—O—C═NR′ (—NR″R′″),

II. Reactive carbonyl groups are exemplified by: aldehydes RCHO andketones RCOR′, acetals RCO(H₂)R′, ketals RR′CO₂R′R″. Reactive carbonylcontaining functional groups known to those well skilled in the art ofprotein immobilization and crosslinking are described in the literature[Pierce Catalog and Handbook, Pierce Chemical Company, Rockford, Ill.(1994); S. S. Wong, Chemistry of Protein Conjugation and Cross-Linking,CRC Press, Boca Raton, Fla. (1991)].

III. Alkyl or aryl donors are exemplified by: alkyl or aryl halidesR-Hal, azides R—N₃, sulfate esters RSO₃R′, phosphate esters RPO(OR′₃),alkyloxonium salts R₃O+, sulfonium R₃S+, nitrate esters RONO₂, Michaelacceptors RCR′═CR′″COR″, aryl fluorides ArF, isonitriles RN+=C−,haloamines R₂N-Hal, alkenes and alkynes.

IV. Sulfur containing groups are exemplified by disulfides RSSR′,sulfhydryls RSH, epoxides R₂C_(—) ^(O)CR′₂.

V. Salts are exemplified by alkyl or aryl ammonium salts R₄N+,carboxylate RCOO—, sulfate ROSO₃—, phosphate ROPO₃″ and amines R₃N.

Table 1 below includes examples of triggers, organized by releasemechanism. In Table 1, R═ is a multifunctional crosslinking agent thatcan be an alkyl, aryl, or other chains with activating groups that canreact with the protein to be crosslinked. Those reactive groups can beany variety of groups such as those susceptible to nucleophilic, freeradical or electrophilic displacement including halides, aldehydes,carbonates, urethanes, xanthanes, epoxides among others.

TABLE 1 Release Trigger Examples Conditions 1. Acid Labile R-O-R H⁺ orLewis Linkers e.g. Thp, MOM, Acidic catalysts Acetal, ketal Aldol,Michael adducts, esters 2. Base Labile R′OCO2-R′ Variety of basicLinkers Carbonates media R′O-CONR₂ Carbamates R₂′NCONR₂ Urethanes Aldol,Michael adducts, esters 3. Fluoride R-OSiR₃ Aqueous F⁻ Labile LinkersVarious Si containing linkers 4. Enzyme RCOOR, RCONR₂′ Free lipases,Labile Linkers amidases, esterases 5. Reduction Disulfide H₂ catalyst;Labile Linkers linkers that Hydrides cleave via Hydrogenolysis ReductiveElimination R′-S-S-R 6. Oxidation R-OSiR₃ Oxidizing Labile LinkersGlycols R- agents: e.g. CH(OH)—CH(OH)—R′ H₂O₂, NaOCl, IO₄ ⁻ Metal basedoxidizers, other hypervalent oxidents 7. Thio-labile R′-S-S-R Thiols,e.g., linkers Cys, DTT, mercaptoethanol 8. Heavy Metal Various AllylTransition metal Labile Linkers Ethers based reagents ROCH₂CH═CHR (Pd,Ir, Hg, Ag, Alkyl, Acyl Cu, Tb Rh) Allyl ester Pd (0) catalysts 9.Photolabile O-nitrobenzyl light (hv) Linkers (ONB) DESYL groups inlinker 10. Free Thiohydroxa- Free radical Radical Labile mate esterinitiator Linkers (Barton ester) 11. Metal- Iron (III) Metal removalchelate linked diphenanthroline e.g. by chelation or precipitation 12.Thermally Peroxides Increase in Labile Linkers R-OO-R temperature 13.“Safety Methylthio- Base; amines, Catch” Labile ethyl (Mte) othersLinkers Dithianes

Additional examples of reversible crosslinkers are described in T. W.Green, Protective Groups in Organic Synthesis, John Wiley & Sons (Eds.)(1981). Any variety of strategies used for reversible protecting groupscan be incorporated into a crosslinker suitable for producingcrosslinked protein crystals capable of reversible, controlledsolubilization. Various approaches are listed, in Waldmann's review ofthis subject, in Angewante Chemie Inl. Ed. Engl., 35, p. 2056 (1996).

Other types of reversible crosslinkers are disulfide bond-containingcrosslinkers. The trigger breaking crosslinks formed by suchcrosslinkers is the addition of reducing agent, such as cysteine, to theenvironment of the crosslinked protein crystals.

Disulfide crosslinkers are described in the Pierce Catalog and Handbook(1994-1995) and more recently in “Bioconjugate Techniques”, By G. T.Hermanson, (1996), Academic Press, Division of Harcourt Brace & Company,525 B Street, Suite 1900, San Diego, Calif. 92101-4495.

Examples of Such Crosslinkers Include:

Homobifunctional (Symmetric)

DSS—Dithiobis(succinimidylpropionate), also know as Lomant's Reagent

DTSSP—3-3′-Dithiobis(sulfosuccinimidylpropionate), water soluble versionof DSP

DTBP—Dimethyl 3,3′-dithiobispropionimidate.HCl

BASED—Bis-(β-[4-azidosalicylamido]ethyl)disulfide

DPDPB—1,4-Di-(3′-[2′-pyridyldithio]-propionamido)butane.

Heterobifunctional (Asymmetric)

SPDP—N-Succinimidyl-3-(2-pyridyldithio)propionate

LC-SPDP—Succinimidyl-6-(3-[2-pyridyldithio] propionate)hexanoate

Sulfo-LC-SPDP—Sulfosuccinimidyl-6-(3-[2-pyridyldlthio]propionate)hexanoate,water soluble version of LC-SPDP

APDP—N-(4-[p-azidosalicylamido]butyl)-3′-(2′-pyridyldithio) propionamide

SADP—N-Succinimidyl(4-azidophenyl)1,3′-dithiopropionate

Sulfo-SADP—Sulfosuccinimidyl(4-azidophenyl) 1,3′-dithiopropionate, watersoluble version of SADP

SAED—Sulfosuccinimidyl-2-(7-azido-4-methycoumarin-3-acetamide)ethyl-1,3'dithiopropionate

SAND—Sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)ethyl-1,3′-dithiopropionate

SASD—Sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3′-dithiopropionate

SMPB—Succinimidyl-4-(p-maleimidophenyl)butyrate

Sulfo-SMPB—Sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate

SMPT—4-Succinimidyloxycarbonyl-methyl-α-(2-pyridylthio) toluene

Sulfo-LC-SMPT—Sulfosuccinimidyl-6-(α-methyl-α-(2-pyridylthio)toluamido)hexanoate.

In particular, see Part II, Chapters 3-5 on Zero-length Cross-linkers,Homobifunctional Cross-linkers and Heterobifunctional Cross-linkers in“Bioconjugate Techniques”, By G. T. Hermanson, (1996), Academic Press,Division of Harcourt Brace & Company, 525 B Street, Suite 1900, SanDiego, Calif. 92101-4495.

Crosslinked protein crystals useful in the protein formulations of thepresent invention may also be prepared according to the methods setforth in PCT patent application PCT/US91/05415.

Encapsulation of Protein Crystals in Polymeric Carriers

According to one embodiment of this invention, compositions are producedwhen protein crystals are encapsulated in at least one polymeric carrierto form microspheres by virtue of encapsulation within the matrix of thepolymeric carrier to preserve their native and biologically activetertiary structure. The crystals can be encapsulated using variousbiocompatible and/or biodegradable polymers having unique propertieswhich are suitable for delivery to different biological environments orfor effecting specific functions. The rate of dissolution and,therefore, delivery of active protein is determined by the particularencapsulation technique, polymer composition, polymer crosslinking,polymer thickness, polymer solubility, protein crystal geometry anddegree and, if any, of protein crystal crosslinking

Protein crystals or formulations to be encapsulated are suspended in apolymeric carrier which is dissolved in an organic solvent. The polymersolution must be concentrated enough to completely coat the proteincrystals or formulations after they are added to the solution. Such anamount is one which provides a weight ratio of protein crystals topolymer between about 0.02 and about 20, preferably between about 0.1and about 2. The protein crystals are contacted with polymer in solutionfor a period of time between about 0.5 minutes and about 30 minutes,preferably between about 1 minutes and about 3 minutes. The crystalsshould be kept suspended and not allowed to aggregate as they are coatedby contact with the polymer.

Following that contact, the crystals become coated and are referred toas nascent microspheres. The nascent microspheres increase in size whilecoating occurs. In a preferred embodiment of the invention, thesuspended coated crystals or nascent microspheres along with thepolymeric carrier and organic solvent are transferred to a larger volumeof an aqueous solution containing a surface active agent, known as anemulsifier. In the aqueous solution, the suspended nascent microspheresare immersed in the aqueous phase, where the organic solvent evaporatesor diffuses away from the polymer. Eventually, a point is reached wherethe polymer is no longer soluble and forms a precipitated phaseencapsulating the protein crystals or formulations to form acomposition. This aspect of the process is referred to as hardening ofthe polymeric carrier or polymer. The emulsifier helps to reduce theinterfacial surface tension between the various phases of matter in thesystem during the hardening phase of the process. Alternatively, if thecoating polymer has some inherent surface activity, there may be no needfor addition of a separate surface active agent.

Emulsifiers useful to prepare encapsulated protein crystals according tothis invention include poly(vinyl alcohol) as exemplified herein,surfactants and other surface active agents which can reduce the surfacetension between the polymer coated protein crystals or polymer coatedcrystal formulations and the solution.

Organic solvents useful to prepare the microspheres of the presentinvention include methylene chloride, ethyl acetate, chloroform andother non-toxic solvents which will depend on the properties of thepolymer. Solvents should be chosen that solubilize the polymer and areultimately non-toxic.

A preferred embodiment of this invention is that the crystallinity ofthe protein crystals is maintained during the encapsulation process. Thecrystallinity is maintained during the coating process by using anorganic solvent in which the crystals are not soluble. Subsequently,once the coated crystals are transferred to the aqueous solvent, rapidhardening of the polymeric carrier and sufficient coating of thecrystals in the previous step shields the crystalline material fromdissolution. In another embodiment, the use of crosslinked proteincrystals facilitates maintenance of crystallinity in both the aqueousand organic solvents.

The polymers used as polymeric carriers to coat the protein crystals canbe either homo-polymers or co-polymers. The rate of hydrolysis of themicrospheres is largely determined by the hydrolysis rate of theindividual polymer species. In general, the rate of hydrolysis decreasesas follows:polycarbonates>polyesters>polyurethanes>polyorthoesters>polyamides. Fora review of biodegradable and biocompatible polymers, see W. R. Gombotzand D. K. Pettit, “Biodegradable polymers for protein and peptide drugdelivery”, Bioconjugate Chemistry, vol. 6, pp. 332-351 (1995).

In a preferred embodiment, the polymeric carrier is composed of a singlepolymer type such as PLGA. In a next preferred embodiment, the polymericcarrier can be a mixture of polymers such as 50% PLGA and 50% albumin.

Other polymers useful as polymeric carriers to prepare encapsulatedprotein crystals according to this invention includebiocompatinie/biodegradable polymers selected from the group consistingof poly (acrylic acid), poly (cyanoacrylates), poly (amino acids), poly(anhydrides), poly (depsipeptide), poly (esters), such as poly (lacticacid) or PLA, poly (b-hydroxybutryate), poly (caprolactone) and poly(dioxanone); poly (ethylene glycol), poly (hydroxypropyl)methacrylamide,poly [(organo)phosphazene], poly (ortho esters), poly (vinyl alcohol),poly (vinylpyrrolidone), maleic anhydride-alkyl vinyl ether copolymers,pluronic polyols, albumin, alginate, cellulose and cellulosederivatives, collagen, fibrin, gelatin, hyaluronic acid,oligosaccharides, glycaminoglycans, sulfated polysaccharides, blends andcopolymers thereof. Other useful polymers are described in J. Heller andR. W. Balar, “Theory and Practice of Controlled Drug Delivery fromBiodegradable Polymers,” Academic Press, New York, N.Y., (1980); K. O.R. Lehman and D. K. Dreher, Pharmaceutical Technology, vol. 3, pp. 5-_,(1979); E. M. Ramadan, A. El-Helw and Y. El-Said, Journal ofMicroencapsulation, vol. 5, p. 125 (1988). The preferred polymer willdepend upon the particular protein component of the protein crystalsused and the intended use of the encapsulated crystals (formulations andcompositions). Alternatively, the solvent evaporation technique may beused for encapsulating protein crystals (see D. Babay, A. Hoffmann andS. Benita, Biomaterials vol. 9, pp. 482-488 (1988).

In a preferred embodiment of this invention, protein crystals areencapsulated in at least one polymeric carrier using a double emulsionmethod, as illustrated herein, using a polymer, such aspolylactic-co-glycolyic acid. In a most preferred embodiment of thisinvention, the polymer is Polylactic-co-glycolyic acid (“PLGA” ). PLGAis a co-polymer prepared by polycondensation reactions with lactic acid(“L”) and glycolic acid (“G” ). Various ratios of L and G can be used tomodulate the crystallinity and hydrophobicity of the PLGA polymer.Higher crystallinity of the polymer results in slower dissolution. PLGApolymers with 20-70% G content tend to be amorphous solids, while highlevel of either G or L result in good polymer crystallinity. For moreinformation on preparing PLGA, see D. K. Gilding and A. M. Reed,“Biodegradable polymers for use in surgery-poly(glycolic)/poly(lacticacid) homo and copolymers: 1., Polymer vol. 20, pp. 1459-1464 (1981).PLGA degrades after exposure to water by hydrolysis of the ester bondlinkage to yield non-toxic monomers of lactic acid and glycolic acid.

In another embodiment, double-walled polymer coated microspheres may beadvantageous. Double-walled polymer coated microspheres may be producedby preparing two separate polymer solutions in methylene chloride orother solvent which can dissolve the polymers. The protein crystals areadded to one of the solutions and dispersed. Here, the protein crystalsbecome coated with the first polymer. Then, the solution containing thefirst polymer coated protein crystals is combined with the secondpolymer solution. [See Pekarek, K. J.; Jacob, J. S. and Mathiowitz, E.Double-walled polymer microspheres for controlled drug release, Nature,367, 258-260]. Now, the second polymer encapsulates the first polymerwhich is encapsulating the protein crystal. Ideally, this solution isthen dripped into a larger volume of an aqueous solution containing asurface active agent or emulsifier. In the aqueous solution, the solventevaporates from the two polymer solutions and the polymers areprecipitated.

The above process can be performed using either protein crystals, DNA orRNA crystals, of formulations of any of these to produce compositions.

Formulations according to this invention comprise a protein crystal,and, at least one ingredient. Such formulations are characterized by atleast a 60 fold greater shelf life when stored at 50° C. than thesoluble form of said protein in solution at 50° C., as measured byT_(½). Alternatively, they are characterized by at least a 59 foldgreater shelf life when stored at 40° C. and 75% humidity than thenonformulated form of said protein crystal when stored at 40° C and 75%humidity, as measured by T_(½). Alternatively, they are characterized byat least a 60% greater shelf life when stored at 50° C. than thenonformulated form of said protein crystal when stored at 50° C, asmeasured by T_(½). Alternatively, they are characterized by the loss ofless than 20% α-helical structural content of the protein after storagefor 4 days at 50° C., wherein the soluble form of said protein losesmore than 50% of its a-helical structural content after storage for 6hours at 50° C, as measured by FTIR. Alternatively, they arecharacterized by the loss of less than 20% α-helical structural contentof the protein after storage for 4 days at 50° C, wherein the solubleform of said protein loses more than 50% of its α-helical structuralcontent after storage for 6 hours at 50° C, as measured by FTIR, andwherein said formulation is characterized by at least a 60 fold greatershelf life when stored at 50° C than the soluble form of said protein insolution at 50° C., as measured by T_(½).

Compositions according to this invention comprise one of the abovedescribed protein crystal formulations, and, at least one polymericcarrier, wherein said formulation is encapsulated within a matix of saidpolymeric carrier.

Alternatively, compositions according to this invention compriseformulations of a protein crystal and at least one ingredient. Suchcompositions may be characterized by at least a 60 fold greater shelflife when stored at 50° C. than the soluble form of said protein insolution at 50° C., as measured by T_(½). Alternatively, they arecharacterized by at least a 59 fold greater shelf life when stored at40° C. and 75% humidity than the nonformulated form of said proteincrystal when stored at 40° C. and 75% humidity, as measured by T_(½).Alternatively, they are characterized by at least a 60% greater shelflife when stored at 50° C. than the nonformulated form of said proteincrystal when stored at 50° C, as measured by T_(½). Alternatively theyare characterized by the loss of less than 20% α-helical structuralcontent of the protein after storage for 4 days at 50° C., wherein thesoluble form of said protein loses more than 50% of its α-helicalstructural content after storage for 6 hours at 50° C., as measured byFTIR. Alternatively, they are characterized by the loss of less than 20%α-helical structural content of the protein after storage for 4 days at50° C., wherein the soluble form of said protein loses more than 50% ofits a-helical structural content after storage for 6 hours at 50° C., asmeasured by FTIR, and wherein said formulation is characterized by atleast a 60 fold greater shelf life when stored at 50° C. than thesoluble form of said protein in solution at 50° C., as measured byT_(½).

In order that this invention may be better understood, the followingexamples are set forth. These examples are for the purpose ofillustration only and are not to be construed as limiting the scope ofthe invention in any manner.

EXAMPLES Example 1 Lipase

Candida rugosa Lipase Crystallization:

Materials:

A—Candida rugosa lipase powder

B—Celite powder (diatomite earth)

C—MPD (2-Methyl-2,4-Pentanediol)

D—5 mM Ca acetate buffer pH 4.6

E—Deionized water

Procedure:

A 1 kg aliquot of lipase powder was mixed well with 1 kg of celite andthen 22 L of distilled water was added. The mixture was stirred todissolve the lipase powder. After dissolution was complete, the pH wasadjusted to 4.8 using acetic acid. Next, the solution was filtered toremove celite and undissolved materials. Then, the filtrate was pumpedthrough a 30 k cut-off hollow fiber to remove all the proteins that wereless than 30 kD molecular weight. Distilled water was added and thelipase filtrate was pumped through the hollow fiber until the retentateconductivity was equal to the conductivity of the distilled water. Atthis point, the addition of distilled water was stopped and 5 mmCa-acetate buffer was added. Next, Ca-acetate buffer was delivered bypumping through the hollow fiber until the conductivity of the retentatewas equal to the conductivity of the Ca-acetate buffer. At that point,addition of the buffer was stopped. The lipase solution was concentratedto 30 mg/ml solution. The crystallization was initiated by pumping MPDslowly into the lipase solution while stirring. Addition of MPD wascontinued until a 20% vol/vol of MPD was reached. The mixture wasstirred for 24 hr or until 90% of the protein had crystallized. Theresulting crystals were washed with crystallization buffer to remove allthe soluble material from the crystals. Then, the crystals weresuspended in fresh crystallization buffer to achieve a proteinconcentration of 42 mg/ml.

Example 2

Formulation of Lipase Crystals Using Sucrose as Excipient:

In order to enhance the stability of lipase crystals during drying andstorage the crystals were formulated with excipients. In this example,lipase crystals were formulated in the slurry form in the presence ofmother liquor before drying. Sucrose (Sigma Chemical Co., St. Louis,Mo.) was added to lipase crystals in mother liquor as an excipient.Sufficient sucrose was added to lipase crystals at a proteinconcentration of 20 mgs/ml in mother liquor (10 mM sodium acetatebuffer, pH 4.8 containing 10 mM Calcium chloride and 20% MPD) to reach afinal concentration of 10%. The resulting suspension was tumbled at roomtemperature for 3 hr. After treatment with sucrose, the crystals wereseparated from the liquid by centrifugation as described in Example 6,method 4 or 5.

Example 3

Formulation of Lipase Crystals Using Trehalose as Excipient:

The lipase crystals were formulated as in Example 2, by addingtrehalose, instead of sucrose, (Sigma Chemical Co., St. Louis, Mo.), toa final concentration of 10% in mother liquor. The resulting suspensionwas tumbled at room temperature for 3 hr and the crystals were separatedfrom the liquid by centrifugation as described in Example 6, method 4 or5.

Example 4

Formulation of Lipase Crystals Using Polyethylene Oxide (PEO) asExcipient:

Lipase crystals were formulated using 0.1% polyethylene oxide in wateras follows. The crystals, in the mother liquor at 20 mg/ml wereseparated from the mother liquor by centrifugation at 1000 rpm in aBeckman GS-6R bench top centrifuge equipped with swinging bucket rotor.Next, the crystals were suspended in 0.1% polyethylene oxide for 3 hrs(Sigma Chemical Co., St. Louis, Mo.) and then separated bycentrifugation, as described in Example 6, method 4 or 5.

Example 5

Formulation of Lipase Crystals Using Methoxypolyethylene Glycol (MOPEG)as Excipient:

Lipase crystals were formulated as in Example 2, by adding 10%methoxypoly ethylene glycol, instead of sucrose, (final concentration)(Sigma Chemical Co., St. Louis, Mo.) in mother liquor and separatingafter 3 hrs by centrifugation, as in Example 6, method 4 or 5.

Example 6

Methods of Drying Crystal Formulations:

Method 1. N₂ Gas Drying at Room Temperature

Crystals as prepared in Examples 1, 10, 14 and 21 were separated fromthe mother liquor containing excipient by centrifugation at 1000 rpm ina Beckman GS-6R bench top centrifuge equipped with swinging bucket rotorin a 50 ml Fisher brand Disposable centrifuge tube (Polypropylene). Thecrystals were then dried by passing a stream of nitrogen atapproximately 10 psi pressure into the tube overnight.

Method 2. Vacuum Oven Drying

Crystals as prepared in Examples 1, 10, 14 and 21 were first separatedfrom the mother liquor/excipient solution using centrifugation at 1000rpm in a Beckman GS-6R bench top centrifuge equipped with swingingbucket rotor in a 50 ml Fisher brand Disposable polypropylene centrifugetube. The wet crystals were then placed in a vacuum oven at 25 in Hg(VWR Scientific Products) at room temperature and dried for at least 12hours.

Method 3. Lyophilization

Crystals as prepared in Examples 1, 10, 14 and 21 were first separatedfrom the mother liquor/excipient solution using centrifugation at 1000rpm in a Beckman GS-6R bench top centrifuge equipped with swingingbucket rotor in a 50 ml Fisher brand Disposable polypropylene centrifugetube. The wet crystals were then freeze dried using a Virtis LyophilizerModel 24 in semistoppered vials. The shelf temperature was slowlyreduced to −40° C. during the freezing step. This temperature was heldfor 16 hrs. Secondary drying was then carried out for another 8 hrs.

Method 4. Organic Solvent and Air Drying

Crystals as prepared in Examples 1, 10, 14 and 21 were first separatedfrom the mother liquor/excipient solution using centrifugation at 1000rpm in a Beckman GS-6R bench top centrifuge equipped with swingingbucket rotor in a 50 ml Fisher brand Disposable polypropylene centrifugetube. The crystals were then suspended in an organic solvent likeethanol or isopropanol or ethyl acetate or other suitable solvents,centrifuged, the supernatant was decanted and air dried at roomtemperature in the fume hood for two days.

Method 5. Air Drying at Room Temperature

Crystals as prepared in Examples 1, 10, 14 and 21 were separated fromthe mother liquor containing excipient by centrifugation at 1000 rpm ina Beckman GS-6R bench top centrifuge equipped with swinging bucket rotorin a 50 ml Fisher brand Disposable centrifuge tube (Polypropylene).Subsequently, the crystals were allowed to air dry in the fume hood fortwo days.

Example 7

Soluble Lipase Sample Preparation:

For comparison, a sample of soluble lipase was prepared by dissolvinglipase crystals to 20 mg/ml in phosphate buffered saline, pH 7.4.

FIG. 2 shows the stability for soluble lipase. Specific activitydecreases extremely rapidly with time. Within 2-3 hours, the specificactivity decreases from about 660 μmoles/min/mg protein to about 100μmoles/min/mg protein or an approximate 85% decrease. The T_(½) forsoluble lipase was calculated to be 1.12 hours.

Example 8

Olive Oil Assay for Measuring Lipase Activity:

Lipase crystals from Examples 1-7 were assessed for activity againstolive oil in pH 7.7 buffer. The assay was carried out titrimetricallyusing slight modifications to the procedure described in PharmaceuticalEnzymes—Properties and Assay Methods, R. Ruyssen and A. Lauwers, (Eds.),Scientific Publishing Company, Ghent, Belgium (1978).

Reagents:

1. Olive oil emulsion:

16.5 gm of gum arabic (Sigma) was dissolved in 180 ml of water, 20 ml ofolive oil (Sigma) and emulsified using a Quick Prep mixer for 3 minutes.

2. Titrant : 0.05 M NaOH

3. Solution A: 3.0 M NaCl

4. Solution B: 75 mM CaCl₂.2H₂O

5. Mix: 40 ml of Solution A was combined with 20 ml of Solution B and100 ml of H₂O.

6. 0.5% Albumin:

7. Lipase Substrate Solution (solution 7) was prepared by adding 50 mlof olive oil emulsion (solution 1) to 40 ml of Mix (solution 5) and 10ml of 0.5% albumin (solution 6).

Assay Procedure

The lipase substrate solution (solution 7) was warmed to 37° C. in awater bath. First, 20 ml of substrate was added to a reaction vessel andthe pH was adjusted to 7.7 using 0.05 M NaOH (solution 2) andequilibrated to 37° C. with stirring. The reaction was initiated byadding enzyme. The reaction progress was monitored by titrating themixture of enzyme and substrate with 0.05 M NaOH to maintain the pH at7.7.

The specific activity (μmoles/min/mg protein) was equal to the initialrate×1000×concentration of the titrant/the amount of enzyme. The zeropoint was determined by running the reaction without enzyme, i.e., usingbuffer in the place of enzyme in the reaction mixture.

Example 9

Activity:

The shelf activity of the dried crystals from Examples 1-5 was measuredusing the olive oil assay as described in Example 8. Dried crystals (5mg) were dissolved in 1 ml of phosphate buffered saline (“PBS”), pH 7.4and the activity was measured using olive oil as substrate.

Shelf Stability:

The shelf stability of dried crystalline lipase formulations fromExamples 2-5 was carried out in a humidity chamber controlled at 75%relative humidity and 40° C. temperature (HOTPACK). The activity of thecrystals was measured by dissolving 5 mg of the dried samples in PBSbuffer, pH 7.4, measuring the activity in the olive oil assay and thencomparing with the initial results.

Crystal Formulations Dried by Method 5

FIG. 3 shows the shelf stability profile of lipase crystals formulatedwith sucrose, trehalose and PEO. When dried by method 5, PEO was themost protective excipient, followed by sucrose and then trehalose.

Crystal Formulations Dried by Method 4

FIG. 4 shows the shelf stability profile of lipase crystals formulatedwith sucrose, trehalose, PEO and MOPEG. When dried by method 4, theexcipients PEO, sucrose and MOPEG were similar in their ability topreserve enzyme activity as measured by their effect on the T_(½).Trehalose was less protective of lipase activity than the otherexcipients.

Shelf Stability

The time required for the specific activity of the enzyme to decrease by50% is known as the T_(½). Table 2 shows the effect on the T_(½) of thespecific activity for formulations of dried lipase crystals. For lipase,sucrose was the most protective excipient, followed by polyethyleneoxide (PEO), methoxypoly ethylene glycol (MOPEG) and finally trehalose.Sucrose was more than 10-fold more protective, as measured by its effecton T_(½) of specific activity.

TABLE 2 Lipase at 40° C. and 75% humidity Dried Lipase CrystalsExcipients T_({fraction (1/2 )})(days) none   1.52 Sucrose 1092Trehalose  90 PEO  835 MOPEG  434 Soluble lipase   0.0468 (1.124 hrs)

The T_(½) was calculated from the shelf life data by non-linearregression analysis using the Sigma Plot program. Table 2 shows thatformulations of lipase were 1,923 fold more stable than soluble when PEOwas used as the excipient. In addition, formulations of lipase were23,300 fold more stable than soluble lipase when sucrose was used as theexcipient (Table 2). Formulations using MOPEG and PEO as excipients withlipase crystals were 9,270 and 17,800 fold more stable than solublelipase (Table 2).

The stability of the formulated crystals relative to the non-formulatedcrystals was greatly enhanced, as shown in Table 2. For example,crystals formulated with trehalose were 59 fold more stable thannon-formulated lipase crystals made without an excipient at 40° C., asshown in Table 2. Similarly, crystals formulated with MOPEG were 286fold more stable than non-formulated crystals and crystals formulatedwith PEO were 549 fold more stable than non-formulated lipase crystalsmade without an excipient at 40° C., as shown in Table 2. Finally,crystals formulated with sucrose were 718 fold more stable thannon-formulated lipase crystals made without an excipient at 40° C., asshown in Table 2.

Moisture Content:

Moisture content was determined by the Karl Fischer method according tomanufacturer's instructions using a Mitsubishi CA-06 Moisture Meterequipped with a VA-06 Vaporizer (Mitsubishi Chemical Corporation, Tokyo,Japan).

TABLE 3 Moisture content lipase crystal formulations % Moisture TIME,DAYS SUCROSE TREHALOSE PEO MOPEG  0  7.23  5.58  8.56  7.03 129 11.1110.45 10.43 10.01

Crystallinity:

The crystal integrity of the formulations were measured by quantitativemicroscopic observations. In order to visualize whether the crystalswere maintained their shape after drying, the dried crystals wereexamined under an Olympus BX60 microscope equipped with DXC-970MD 3CCDColor Video Camera with Camera Adapter (CMA D2) with Image ProPlussoftware. Samples of dried crystals were covered with a glass coverslip,mounted and examined under 10× magnification, using an Olympusmicroscope with an Olympus UPLAN F1 objective lens 10×/0.30 PH1 (phasecontrast).

In this analysis, the crystals originally formulated with sucrose,trehalose, PEO and MOPEG were readily visualized after 129 days at 40°C. and 75% humidity. FIG. 5 shows that crystals formulated with PEO werepresent at the initial time. A similar microscopic observation taken 129days later and shown in FIG. 6 demonstrates that crystallinity wasmaintained for the entire time period. Similar data was obtained forcrystals formulated with sucrose, MOPEG and trehalose (data not shown).

Secondary Structure Characterization by FTIR:

The fourier transform infrared (“FTIR”) spectra were collected on aNicolet model 550 Magna series spectrometer as described by Dong et al.[Dong, A., Caughey, B., Caughey, W. S., Bhat, K. S. and Coe, J. E.Biochemistry, 1992; 31:9364-9370; Dong, A. Prestrelski, S. J., Allison,S. D. and Carpenter, J. F. J.Pharm. Sci., 1995; 84: 415-424.] For thesolid samples, 1 to 2 mg of the protein was lightly ground with 350 mgof KBr powder and filled into small cups used for diffuse reflectanceaccessory. The spectra were collected and then processed using Grams 32from Galactic software for the determination of relative areas of theindividual components of secondary structure using second derivative andcurve-fitting program under amide I region (1600-1700 cm⁻¹).

For comparison, a soluble lipase sample was prepared by dissolvinglipase crystals in phosphate buffered saline and analyzed for stabilityby FTIR.

Secondary structure was determined as follows: FTIR spectra werecollected on a Nicolet model 550 Magna series spectrometer. A 1 mlsample of soluble lipase was placed on a Zinc selenide crystal of ARKESP. The spectra were collected at initial (0) time and after the lossof most of the activity or, near-zero activity. The acquired data wasthen processed using Grams 32 software from Galactic Software for thedetermination of relative areas of the individual components ofsecondary structure using second derivative and curve-fitting programunder amide I region (1600-1700 cmt⁻¹).

TABLE 4 Lipase at 40° C. and 75% Humidity Extended Sample α-Helixβ-Sheets β-Turn coil Random Soluble 25.08 59.89 4.57  3.21 7.25 Lipaseinitial time After 3 13.14 16.11 13.59   6.74 50.42  days at 40° C.Lipase - 19.46 49.94 0.00 30.60 0.00 Sucrose initial time After 66 17.7060.70 0.00 21.60 0.00 days at 40° C. and 75% Humidity Lipase - 23.3052.33 0.00 23.17 1.20 Trehalose initial time After 66 18.38 55.51 0.0026.11 0.00 days at 40° C. and 75% Humidity Lipase-PEO 23.22 48.33 0.0024.53 3.92 initial time After 66 21.79 54.90 1.65 21.66 0.00 days at 40°C. and 75% Humidity Lipase - 24.66 49.37 0.00 19.20 6.77 MOPEG initialtime After 66 26.59 51.09 0.00 22.32 0.00 days at 40° C. and 75%Humidity

Conclusion:

Table 4 shows that for soluble lipase, approximately 50% and 75% of theα-helix and β-sheet structure content was lost over three days. Therewas a corresponding increase in the content of random structure.

In contrast, Table 4 shows that crystals which were formulated and driedwere much more stable than the soluble enzyme. Such crystals showed aloss of a-helical structure which ranged from 6.1% to 21.1% after 66days at 40° C. and 75% humidity. At the same time, random coil insolution increased from 7-50% over 3 days, while crystallinity showedminimum random coil content even after 66 days.

The data in Table 4, obtained using FTIR to monitor changes in secondarystructure, correlated with the activity data shown in FIG. 4. Inparticular, lipase formulated with sucrose, PEO and MOPEG showedsignificantly less loss of α-helical structure and maintained a higherspecific activity over the 66 day time period at elevated temperatureand humidity than crystals formulated with trehalose, which showed a 21%loss of α-helical structure and had the lowest activity profile.

Example 10 Human Serum Albumin

Crystallization of Human Serum Albumin

Ten grams of powdered human serum albumin was added to a 75 ml stirredsolution of 100 mM phosphate buffer (pH 5.5) at 4° C. Final proteinconcentration was 120 mg/ml, estimated from the OD₂₈₀ value of thesolution. First, a saturated ammonium sulfate solution (767 g/l),prepared in deionized water, was added to the protein solution to afinal concentration of 350 g/l or 50% saturation. Next, thecrystallization solution was “seeded” with 1 ml of 50 mg/ml albumincrystals in 50% ammonium sulfate at pH 5.5. Seed crystals were preparedby washing a sample of crystals free of precipitate with a solution of50% saturated ammonium sulfate in 100 mM phosphate buffer at pH 5.5. Theseeded crystallization solution was incubated overnight at 4° C. on avigorously rotating platform. Crystals in the shape of rods (20 μm)appeared in the solution overnight after approximately 16 hr.

Example 11

Formulation of HSA Crystals Using Gelatin as Excipient:

In order to enhance the stability of human serum albumin (HSA) crystalsduring drying and storage the crystals were formulated with excipients.In this example, HSA crystals were formulated in the slurry form in thepresence of mother liquor before drying. Gelatin (Sigma Chemical Co.,St. Louis, Mo.) was added to lipase crystals in mother liquor as anexcipient. Sufficient gelatin was added to lipase crystals at a proteinconcentration 20 mgs/ml in mother liquor (2.5M ammonium sulfate in 100mM phosphate buffer, pH 5.5) to reach a final concentration of 10%. Theresulting suspension was tumbled at room temperature for 3 hr. Aftertreatment with gelatin, the crystals were separated from the liquid bycentrifugation as described in Example 6, method 1.

Drying of HSA Crystals

The HSA crystals were then dried by the four methods described inExample 6. The crystals were suspended in cold (4° C.) ethanol as theorganic solvent of method 4.

Example 12

Soluble Human Serum Albumin Preparation:

For comparison, the soluble HSA sample was prepared by dissolving HSAcrystals at 20 mg/ml in water.

Example 13

Shelf Stability:

Shelf stability of HSA dried crystal formulations were carried out in awaterbath at 50° C. temperature. The stability of the crystals wasmonitored by following structural degradation by FTIR analysis.

Moisture Content:

Moisture content was determined by the Karl Fischer method according tomanufacturer's instructions using a Mitsubishi CA-06 Moisture Meterequipped with a VA-06 Vaporizer (Mitsubishi Chemical Corporation, Tokyo,Japan).

TABLE 5 Moisture content HSA crystals % Moisture TIME, DAYS Gelatin  05.0464 12 6.3841

Crystallinity:

The crystal integrity of the formulations was measured by quantitativemicroscopic observations, as described in Example 9. FIG. 7 shows thatHSA crystals were readily visualized immediately after preparing theformulation with gelatin. FIG. 8 shows that crystallinity was maintainedafter four days at 50° C.

Secondary Structure Characterization by FTIR:

The FTIR spectra were collected on a Nicolet model 550 Magna seriesspectrometer as described by Dong et al.[Dong, A., Caughey, B., Caughey,W. S., Bhat, K. S. and Coe, J. E. Biochemistry, 1992; 31:9364-9370;Dong, A. Prestrelski, S. J., Allison, S. D. and Carpenter, J. F.J.Pharm. Sci., 1995; 84: 415-424]. For the solid samples, 1 to 2 mg ofthe protein was lightly ground with 350 mg of KBr powder and filled intosmall cups used for diffuse reflectance accessory. The spectra werecollected and processed using Grams 32 from Galactic software for thedetermination of relative areas of the individual components ofsecondary structure using a second derivative and curve-fitting programunder amide I region (1600-1700 cm⁻¹).

For comparison, the soluble HSA sample was prepared by dissolving HSAcrystals in water and tested for stability by FTIR. The secondarystructure was determined as in Example 9.

TABLE 6 Secondary Structure content of HSA α- β- Extended Sample HelixSheets β-Turn coil Random HSA- 45.47  6.83 6.79 40.91 0.00 Soluble HSA-28.71 30.39 8.59 28.07 4.24 Sol. 15 min HSA- 19.15 34.69 8.16 27.9110.09  Sol. 30 min HSA- 10.04 15.82 0.00 42.83 31.31  Sol. 60 min HSAcrystals HSA 49.08 24.77 0.00 26.15 0.00 Gelatin HSA 40.93  7.65 7.4843.94 0.00 Gelatin After 12 days at 50° C.

Conclusion:

Soluble HSA showed a rapid 78% decrease in α-helical content after 1hour in solution and a corresponding increase in random coil structure.

Dry formulated crystals showed an approximately 16% decrease inα-helical content, a large decrease in β-sheet content and no increasein the content of random structure.

Example 14 Penicillin Acylase

Crystallization of Penicillin Acylase

An ammonium sulfate suspension of Penicillin acylase from BoehringerMannheim was the raw material. The suspension of Penicillin acylase wasdiluted 1:3 with deionized water. This solution was concentrated bydiafilration using a 30K membrane to a final concentration of 200 OD atA_(280nm)/ml. The enzyme solution was then diluted with 4 M NaH₂PO₄.H₂Oto 150 OD A_(280nm)/ml.

A biphasic solution of 4 M and 1 M sufficient NaH₂PO₄.H₂O to yield afinal solution concentration of 1.9 M NaPO₄, was prepared. In this case,280 ml of 1 M NaH₂PO₄.H₂O was carefully overlaid on top of to the top of549 ml of 4 M NaH₂PO₄.H₂O. The enzyme solution was poured gently intothe side of the container to form a layer above the 1 M layer. Anoverhead stirrer was set up with a marine impeller. The agitator wasplaced into the container with the blades just below the 4M/1Minterface. The speed was adjusted to a setting of 8.0, or 600 rpm. Theagitator was switched on and stopped after 10 minutes. The impeller wasremoved from the container. The volume of seed crystals was measuredwith a graduated pipette using between 0.5 and 0.1% by volume and addedto the 20 L sterile polycarbonate container. The seed was not allowed tobe static for more than 10 seconds before addition. The solution wasmixed by hand using a flat-blade impeller for about 1 minute. Thecrystallization mixture was allowed to stand for 24 hours.

After 24 hours, the container was opened and the solution was mixed byhand for 30 seconds. The solution was allowed to sit for an additional24 hours. After a total of 48 hours, a 10 ml sample was taken of thesupernatant. The sample was filtered with a 0.2 micron Acrodisc. TheA_(280nm) of the supernatant was measured. When supernatant A_(280nm)was greater than 1.0 mg/ml, the crystallization was allowed to continuefor 24 more hours. When the supernatant concentration was less than 1.0mg/ml, the A_(280nm) Of crystal slurry and supernatant directly weredetermined.

Example 15

Penicillin G Assay for PA Crystals

The basis of the activity assay for Penicillin acylase involves atitrimetric assay which measures the enzymatic hydrolysis of benzylpenicillin by the enzyme. The enzyme catalyzes cleavage of a phenylacetyl group from penicillin G thus causing a decrease in pH. Thisactivity was followed by measuring the volume of 50 mM NaOH needed (tomaintain a pH of 8 at 28° C.) per minute of reaction. The assay uses asubstrate buffer made with potassium chloride and Tris and was reportedin U/mg.

Penicillin Acylase Assay:

Chemicals and Solutions Used in the Assay:

1. Penicillin-G, Sigma, Potassium salt

2. 0.05 N Sodium hydroxide

3. 1.0 M KCl, 20 mM Tris buffer, pH 8.0

4. 10 mM Tris, 10 mM CaCl₂ buffer, pH 8.0

5. 0.01 M PBS buffer, pH 7.5

Preparation of Substrate Solution

1 g of penicillin-G was added to 10 ml of 1.0 M KCl, 20 mM Tris buffer,pH 8.0 solution and about 70 ml of DI water, 1 ml of 10 mM Tris, 10 mMCaCl₂. Then the pH of the solution was adjusted to 8.0 using 0.05 NNaOH. Next, final solution volume was adjusted to 100 ml. The solutionwas prepared fresh before each use and used within five hours ofpreparation.

Preparation of PA Sample Solution

A sample of Penicillin acylase was dissolved in PBS buffer, pH 7.4.Typically, 2 to 4 mg (dry weight) of protein were used for each assay.

Assay for Hydrolysis of Penicillin G

20 ml of penicillin acylase substrate solution was added to a titrationvessel and equilibrated to 28° C. After adding the enzyme solution tothe reaction vessel, the hydrolysis of penicillin-G was monitored bytitrating the reaction mixture with 0.05 N NaOH to maintain the pH at8.0.

Example 16

Formulation of PA Crystals Using Hydroxypropyl-β-cyclodextrin (HPCD) asExcipient:

In order to enhance the stability of PA crystals during drying andstorage, the crystals were formulated with excipients. In this example,PA crystals were formulated in the slurry form in the presence of motherliquor before drying. Hydroxypropyl-β-cyclodextrin (HPCD) (SigmaChemical Co., St. Louis, Mo.) was added to PA crystals in mother liquoras an excipient. Sufficient HPCD was added to lipase crystals at aprotein concentration 20 mgs/m in mother liquor (1.9M NaH₂PO₄, pH 6.6),to reach a final concentration of 10%. The resulting suspension wastumbled at room temperature for 3 hr. After treatment with HPCD, thecrystals were separated from the liquid by centrifugation as describedin Example 6, method 1.

Example 17

Formulation of PA Crystals Using Mother Liquor Itself as Excipient:

The PA crystals were formulated using mother liquor (1.9M NaH₂PO₄, pH6.6). The crystals were separated by centrifugation as in Example 16.

Example 18

Drying PA

The PA crystals from Examples 15-17 were then dried according to themethods 1, 3 or 4 of Example 6. The organic solvent used for method 4 ofExample 6 was ethyl acetate.

Example 19

Soluble PA Preparation:

As a standard of comparison, a sample of soluble PA was prepared bydissolving PA crystals in water at 20 mg/ml.

The stability of soluble PA was measured over time and a plot specificactivity at 55° C. versus time is shown in FIG. 9. PA enzyme activity insolution decayed rapidly and was undetectable after approximately 20hours.

Example 20

Activity of the Dried Crystals:

The activity of the dried crystals from Example 18 was tested in the PenG assay as described in Example 15. The dried crystals (5 mg) weredissolved in 1 ml of water and the activity was measured using Pen G assubstrate

Shelf Stability:

The shelf stability of PA dried crystal formulations was determinedusing a Reactive-Therm III—Heating/Stirring module by Pierce at 55° C.temperature. Activities were measured by dissolving 5 mg of the driedsample in water and measuring the enzyme activity in the Pen G assaydescribed in Example 15 and compared with the initial results. FIG. 10depicts the shelf stability profiles of PA crystals with and withoutexcipient. FIG. 10 also depicts shelf stability profiles of PA crystalformulations dried with nitrogen (method 1) or air dried (method 4).

TABLE 7 Penicillin acylase activity at 55° C. Dry Crystals-Penicillinacylase T{fraction (1/2 )}days Penicillin acylase - crystals dried by123.25 lyophilization Penicillin acylase - HPCD dried by  99.24lyophilization Penicillin acylase - dried using  24.85 nitrogenPenicillin acylase - HPCD dried by  87.91 nitrogen Soluble Penicillinacylase  0.21 (4.92 hrs)

The T_(½) was calculated from the shelf life data by non-linearregression analysis using the Sigma Plot program. The stability of theformulated crystals relative to the non-formulated crystals wasenhanced, as shown in Table 7. For example, crystals formulated withHPCD were 418 fold more stable soluble PA at 55° C. (Table 7). Crystalsformulated with HPCD were 3.5 fold more stable at 55° C. thannon-formulated PA crystals made without an excipient, as shown in Table7.

Moisture Content:

Moisture content was determined by the Karl Fischer method according tomanufacturer's instructions using a Mitsubishi CA-06 Moisture Meterequipped with a VA-06 Vaporizer (Mitsubishi Chemical Corporation, Tokyo,Japan).

TABLE 8 Moisture content of PA crystals % Moisture DAYS PA HPCD  02.6198 1.5722 43 2.1792 3.1963

Crystallinity:

The crystal integrity of the formulations was measured by quantitativemicroscopic observations as described in Example 9. Crystallinity wasmaintained through out the process as indicated by the crystals, whichwere readily visible (data not shown).

Secondary Structure Characterization by FTIR:

Stability was assessed by quantifying the secondary stucture content ofthe dried and formulated PA crystals by FTIR as described in Example 9.Soluble PA was used for comparative purposes.

TABLE 9 Secondary Structure of PA at 55° C. Extended Sample α-Helixβ-Sheets β-Turn coil Random Soluble PA 37.74 33.53 11.57 17.16  0.00Init. time After 2 25.27 35.32  7.29 21.93 10.19 hrs at 55° C. After 9.29 50.66  0.00 26.77 13.28 24 hrs at 55° C. PA - dried 33.83 27.99 5.32 13.66  0.00 by lyoph. init. time After 22.53 18.9  17.03 18.4223.12 43 days at 55° C. PA - HPCD 35.9  36.96  0.00 27.14  0.00 dried bylyoph. init. time After 19.05 16.77 23.11 16.65 24.42 43 days at 55° C.PA - dried 25.73 34.93  6.68 28.73  3.93 by N2 init. time no excipientAfter 11.88 34.73 10.22 30.07 13.10 43 days at 55° C. PA - HPCD 31.2635.78 12.24 20.72  0.00 dried by nitrogen init. time After 22.17 25.310.00 42.23 10.29 43 days at 55° C.

Conclusion:

The loss of α-helical content was 76% after 1 day for soluble Penicillinacylase. In contrast, when HPCD was used as an excipient and theformulation was dried by method 1 of Example 6, only 31% of theα-helical content was lost after 43 days at 55° C. temperature.

Example 21 Glucose Oxidase

Preparation of Glucose Oxidase Crystals

We prepared crystals of glucose oxidase as follows. First, theglycoprotein was purified by anion exchange chromatography and then thecrystallization parameters were optimized (data not shown).

As a result of these studies, we found that the conditions forcrystallization of glucose oxidase fall generally in the range of 7 to17% PEG 4000 or 6000, 8 to 20% 2-propanol or ethanol and buffer toadjust the pH to between 3 and 6. It should be understood, however, thatmany sets of experimental conditions within and near this range canproduce satisfactory results for the crystallization of glucose oxidaseand other glycoproteins. Those of skill in the art will appreciate thatthe precise conditions which efficiently produce crystals of the desiredsize and quality, will vary due to differences in experimentalconditions, such as protein and reagent purity, rates of stirring, shearforce effects and carbohydrate content.

Large Scale Crystallization of Glucose Oxidase

We determined preferred conditions for preparative scale crystallizationof glucose oxidase. Preparative scale crystallization generally involves100 to 900 mls of glycoprotein.

A. Crystallization at Constant pH Without Seed

Glucose oxidase was diafiltered in water and concentrated to an A₂₈₀ ofbetween 5 and 15. The glucose oxidase concentrate was mixed (1:1) withone volume of the crystallizing reagent containing 18% PEG 6000, 32%2-propanol in 0.2 M Na-Acetate at pH 5.0. After mixing, the solution wascooled to 6° C. The glucose oxidase crystallization solution was stirredfor 24 hours at 100 rpm with a propeller stirrer. During this time, thecrystals formed gradually.

Example 22

Formulation of Glucose Oxidase Crystals Using Trehalose as Excipient:

In order to enhance the stability of glucose oxidase (GOD) crystalsduring drying and storage, the crystals were formulated with excipients.In this example, GOD crystals were formulated in the slurry form in thepresence of mother liquor before drying. Trehalose (Sigma Chemical Co.,St. Louis, Mo.) was added to GOD crystals in mother liquor as anexcipient. Sufficient trehalose was added to GOD crystals at a proteinconcentration 20 mgs/ml in mother liquor (100 mM sodium acetate buffer,pH 5.5 containing 32% isopropanol and 9% PEG 6000) to reach a finalconcentration of 10%. The resulting suspension was tumbled at roomtemperature for 3 hr. After treatment with trehalose, the crystals wereseparated from the liquid by centrifugation as described in Example 6,method 1.

Example 23

Formulation of Glucose Oxidase Crystals Using Lactitol as Excipient:

Glucose oxidase crystals were formulated as in Example 22 by addinglactitol (Sigma Chemical Co. St. Louis, Mo.), (instead of trehalose) toa final concentration of 10% to the mother liquor. The crystals wereseparated from the mother liquor/lactitol solution after three hours bycentrifugation.

Example 24

Formulation of Glucose Oxidase Crystals UsingHydroxypropyl-β-cyclodextrin (HPCD) as Excipient:

Glucose oxidase crystals were formulated usinghydroxypropyl-β-cyclodextrin (HPCD) as in Example 22 (instead oftrehalose) by adding HPCD to a final concentration of 10% in motherliquor and incubated for 3 hrs (Sigma Chemical Co. St. Louis, Mo.). Thecrystals were then separated from the mother liquor/HPCD solution after3 hr. by centrifugation as described in Example 6, method 1.

Example 25

Formulation of Glucose Oxidase Crystals Using Gelatin as Excipient:

Glucose oxidase crystals were formulated as in Example 22 by addinggelatin to a final concentration of 10% (Sigma Chemical Co. St. Louis,Mo.) in mother liquor (instead of trehalose). The crystals wereseparated from the mother liquor/gelatin solution after three hours bycentrifugation.

Example 26

Formulation of Glucose Oxidase Crystals Using Methoxypolyethylene Glycolas Excipient:

The glucose oxidase crystals were formulated as in Example 22 by addingmethoxypoly ethylene glycol to a final concentration of 10% (SigmaChemical Co. St. Louis, Mo.) in mother liquor. The crystals wereseparated from the mother liquor/methoxypoly ethylene glycol solutionafter 3 hrs by centrifugation as described in Example 6, method 1.

Example 27

Formulation of Glucose Oxidase Crystals Using Sucrose as Excipient:

Glucose oxidase crystals were formulated as in Example 22 by addingsucrose to a final concentration of 10% (Sigma Chemical Co., St. Louis,Mo.) in the mother liquor. The crystals were separated from the motherliquor/sucrose solution after three hours by centrifugation.

Example 28

Drying Glucose Oxidase Formulations

The glucose oxidase crystal formulations described above were driedaccording to the methods described in Example 6. Method 4 utilized cold(4° C.) isopropanol as the organic solvent.

Example 29

Soluble Glucose Oxidase Preparation:

For comparison, the soluble glucose oxidase sample was prepared bydissolving glucose oxidase crystals at 20 mg/ml in 50 mM citrate buffer,pH 6.0.

FIG. 11 shows the stability of the soluble glucose oxidase over time at50° C. The specific activity declines rapidly with time. After 24 hours,the specific activity decreases by more than 90%. The T_(½) for solubleglucose oxidase was calculated to be 0.91 hours.

Example 30

Glucose Oxidase Activity Assay:

The following protocol was used to determine the activity of driedglucose oxidase crystals and crystal formulations.

Chemicals and solutions:

1. Phosphate buffer (20 mM, pH 7.3), NaCl (0.1 M) solution,

2. 21 mM O-Dianisidine dihydrochloride stock solution, diluted to 0.21mM as working solution before use,

3. 2 M glucose solution,

4. Peroxide solution (2 mg/ml),

5. 50 mM citrate buffer, pH 6.0

Sample Preparation:

1. Add 2 mg of glucose oxidase to 1 ml of 50 mM citrate buffer, pH 6.0and vortexed well for about 2 min. The solution was mixed by tumbling atroom temperature for 1 hour to reconstitute.

2. Prepare a dilute enzyme solution by mixing 0.1 ml of the above enzymesolution with 4.9 ml of the same citrate buffer.

Assay Procedure for Enzyme Activity Measurement:

1. The assay was monitored by a UV-Vis spectrophotometer. Use thekinetic mode and set wavelength at 460 nm and temperature at 25° C.

2. Warm up the 0-dianisidine/phosphate working solution in the 25° C.water bath and bubble the solution with oxygen for at least 20 min.before use.

3. Measure the blank using the reagent solution without the enzymesolution added.

4. Pipette 2.4 ml of oxygenated O-dianisidine/phosphate workingsolution, 0.4 ml of 2 M glucose solution and 0.1 ml of peroxidase into adisposable cuvette.

5. Add 10 ml of the enzyme sample on the cuvette wall (tilt the cuvetteto prevent the sample from mixing with the reagent at this step) andcover with a piece of parafilm. Mix quickly by inverting the cuvettetwice, insert the cuvette into the spectrophotometer's cell compartmentand start to collect data.

Calculate the enzyme specific activity using the following formula

Specific activity=A*B*C/D*E*F

Where:

A=The changed in units of absorbance at 460 nm per minute

B=Reaction mixture volume (ml)

C=Dilution factor

D=11.3 (a constant)

E=Weight (mg) of the enzyme used

F=Sample volume (ml)

Example 31

Activity of the Dried Crystals:

The activity of the dried crystal formulations of glucose oxidase wasmeasured as described in Example 30.

Shelf Stability:

A study of the shelf stability of formulations of glucose oxidasecrystals was performed. In this case, the formulations were dried bymethod 4 of Example 6 and were stored in a 2 ml screw cap Eppendorf tubein a waterbath at 50° C. temperature for 13 days. Activities at specifictime points were obtained by dissolving 2 mg of the dried sample in 50mM citrate buffer, pH 6.0 and then measuring the activity according toExample 30.

The shelf stabilities of the various glucose oxidase formulations storedat 50° C. were determined. The data are presented in FIG. 12. Lactitolwas the most effective excipient at preserving glucose oxidase specificactivity over time at elevated temperature.

TABLE 10 Glucose Oxidase at 50° C. Dried glucose oxidase crystalsExcipient T_(½) days none  1.52 Trehalose  3.8 Lactitol  4.85 HPCD  2.4MOPEG 13.1 Gelatin  3.95 Sucrose  3.27 Soluble GOD  0.04 (0.91 hrs)

The T_(½) was calculated from the shelf life data by non-linearregression analysis using the Sigma Plot program. Table 10 shows thatformulations of glucose oxidase were 95 fold more stable than solublewhen trehalose was used as the excipient. In addition, formulations ofglucose oxidase were 121 fold more stable than soluble when lactitol wasused as the excipient (Table 10). Formulations using HPCD or MOPEG asexcipients with glucose oxidase crystals were 60 and 325 fold morestable than soluble glucose oxidase, respectively(Table 10). Finally,formulations using gelatin or sucrose as excipients with glucose oxidasecrystals were 99 and 82 fold more stable than soluble, respectively(Table 10).

Formulations made with either trehalose, lactitol, HPCD, MOPEG, gelatinor sucrose, as excipients with glucose oxidase crystals were 2.5 fold,3.2 fold, 1.6 fold, 8.6 fold, 2.6 fold, or 2.2 fold more stable thanglucose oxidase crystals made without an excipient at 50° C., asmeasured by T_(½), as shown in Table 10.

Moisture Content:

Moisture content was determined by the Karl Fischer method according tomanufacturer's instructions using a Mitsubishi CA-06 Moisture Meterequipped with a VA-06 Vaporizer (Mitsubishi Chemical Corporation, Tokyo,Japan).

TABLE 11 Moisture content of GOD crystal formulations TIME % MoistureDAYS Trehalose Lactitol HPCD MOPEG Gelatin Sucrose 0 4.3819 4.52748.3817 4.2008 4.7090 4.2083 13 8.7292 11.4808 8.7582 8.0763 13.75419.8284

Crystallinity:

The crystal integrity of the GOD formulations was measured byquantitative microscopic observations as described in Example 9. In thisexample, the crystals were readily visualized, indicating thatcrystallinity was maintained throughout the process.

FIG. 13 show that glucose oxidase crystals were readily visualizedimmediately after preparing the lactitol formulation. FIG. 14demonstrates that crystallinity was maintained after 13 days at 50° C.Crystalline material was also readily visualized after formulatingglucose oxidase with trehalose, as depicted in FIG. 15. Likewise,crystalline material remained readily visualized after after 13 days at50° C., as shown in FIG. 16.

Secondary Structure Characterization by FTIR:

Stability was assessed by quantifying the secondary stucture content ofthe dried and formulated GOD crystals by FTIR as described in Example 9.For comparison, a soluble glucose oxidase sample was prepared bydissolving glucose oxidase crystals in 50 mM citrate buffer at pH 6.0and placing about 1 ml on a Zinc selenide crystal of ARK ESP, which thenanalyzed for stability by FTIR.

TABLE 12 Soluble Glucose Oxidase at 50° C. Extended Sample α-Helixβ-Sheets β-Turn coil Random Soluble GOD 35.65 29.14 14.02 8.32 12.87init. time After 1 hr 23.05 34.78 6.17 18.56 17.44 at 50° C. After 6 hr9.92 25.45 8.44 32.04 24.15 at 50° C. GOD-Trehalose 30.34 29.10 8.2823.13 9.15 init. time After 4 days 24.63 33.32 5.78 27.17 9.10 at 50° C.GOD-Lactitol 33.82 25.88 8.82 20.41 11.07 init. time After 4 days 27.2427.53 8.56 19.82 16.85 at 50° C. GOD-HPCD 29.81 25.75 8.46 23.10 12.88init. time After 4 days 17.15 15.75 6.12 31.67 29.31 at 50° C. GOD-MOPEG25.45 28.23 9.62 22.52 14.18 init. time After 4 days 15.87 30.91 8.9128.68 15.63 at 50° C. GOD-gelatin 31.05 34.05 7.05 23.13 4.72 init. timeAfter 4 days 23.49 26.2 8.58 16.24 25.49 at 50° C. GOD-Sucrose 30.1229.68 11.03 19.75 9.42 init. time After 4 days 24.68 23.75 9.58 31.5310.46 at 50° C.

Conclusion:

Soluble glucose oxidase lost 75% of its α-helical content within only 6hours at 50° C.

The sugars lactitol, sucrose and trehalose were the most effectiveexcipients in preventing loss of α-helical content upon storage at anelevated temperature. Glucose oxidase crystals formulated in lactitol,sucrose and trehalose and dried by method 4 showed only an 18.1-19.4 %loss of α-helical structural content after 4 days at 50° C.

Example 32

Drying of Candida Rugosa Lipase Crystals:

Materials:

A—Candida rugosa lipase (Example 1)

B—Poly(ethylene glycol), 100% PEG 200, 300, 400, or 600

C—acetone

Procedure:

A 4 ml aliquot of crystal suspension (140 mg) is added to four 15 mltubes. Next, the suspension is centrifuged at between 1000 to 3000 RPMfor between 1 to 5 minutes or until the crystallization buffer isremoved. Then, 4 ml of liquid polymer (any PEG between 200 to 600 issuitable) is added to each tube and the contents are mixed untilhomogeneous. The suspension is centrifuged at between 1000 to 3000 RPMfor between 1 to 5 minutes or until the liquid polymer is removed. Next,4 ml of acetone (isopropanol, butanol and other solvents are alsosuitable) is added to each tube and mixed well. The crystal/organicsolvent suspensions are transferred to 0.8 cm×4 cm BIO-RAD poly-prepchromatography columns (spin columns). The columns are centrifuged at1000 RPM for 1 to 5 minutes to remove the organic solvent. Finally,nitrogen gas is passed through the column to dry the crystals until afree flowing powder results.

Example 33

Purafect (protease) 4000 L Crystallization:

Materials:

A—crude purafect 4000 L

B—15% NaSO₄ solution

Procedure:

One volume of crude purafect enzyme solution is mixed with two volumesof 15% Na₂SO₄ solution. The mixture is stirred for 24 hr at roomtemperature or until the crystallization is completed. The crystals arewashed with 15% Na₂SO₄ solution to eliminate the soluble enzyme. Thecrystals are suspended in fresh 15% Na₂SO₄ solution to yield a proteinconcentration of 27 mg/ml.

Example 34

Drying of Purafect Crystals:

Materials:

A—purafect crystals suspension

B—Poly(ethylene glycol), 100% PEG 200, 300, 400, or 600

C—organic solution

Procedure:

A 4 ml aliquot of crystal suspension (140 mg) is added to four 15 mltubes. Next, the suspension is centrifuged at between 1000 to 3000 RPMfor between 1 to 5 minutes or until the crystallization buffer isremoved. Then, 4 ml of liquid polymer (any PEG between 200 to 600 issuitable) is added to each tube and the contents are mixed untilhomogeneous. The suspension is centrifuged at between 1000 to 3000 RPMfor between 1 to 5 minutes or until the liquid polymer is removed. Next,4 ml of acetone (isopropanol, butanol and other solvents are alsosuitable) is added to each tube and mixed well. The crystal/organicsolvent suspensions are transferred to 0.8 cm×4 cm BIO-RAD poly-prepchromatography columns (spin columns). The columns are centrifuged at1000 RPM for 1 to 5 minutes to remove the organic solvent. Finally,nitrogen gas is passed through the column to dry the crystals until afree flowing powder results.

Example 35

Producing DNA for Crystallization

Plasmids derived from pUC plasmids, such as pSP64, may be used toproduce either DNA or mRNA for crystallization. In this example, plasmidpSP64 (available from Promega Biological Research Products) is used togenerate DNA for crystallization. The cDNA coding for the protein ofinterest is inserted into any of a number restriction sites available inthe multiple cloning site. The recombinant plasmid is then used totransform E. coli bacteria. Next, large amounts of plasmid are obtainedby growth of the bacteria in ampicillin containing medium. Thetechniques for producing the recombinant plasmid, transforming E. colicells and for bacterial growth and plasmid DNA preparation are describedin detail in “Molecular Cloning, 2nd Edition” (1989) Sambrook, J.,Fritsch, E. F. and T. Maniatis.

Plasmid DNA is subsequently purified from the bacterial cultures byfirst lysing the cells and then separating the plasmid DNA from thegenomic DNA, RNA and other cellular materials using CsCl gradients.These techniques are well known in the art and are discussed in detailin Sambrook et al. The gradient purified DNA is extracted with Tris-EDTAbuffer saturated N-butanol and finally ethanol. Next, plasmid DNA issubjected to either linearization of the plasmid, for use in thegeneration of mRNA for RNA crystallization (Example 36) or excision ofthe gene of interest for DNA crystallization (Example 37).

Example 36

Producing mRNA for Crystallization

The SP64 plasmid in combination with SP6 RNA Polymerase (available fromPromega Biological Research Products) are used for the generation ofmilligram quantities of 5′ capped RNA transcripts. The plasmid preparedin Example 35, prior to excision of the gene, is linearized with arestriction enzyme downstream of the poly A tail. The linear plasmid ispurified by 2 phenol/chloroform and 2 chloroform extractions. DNA isnext precipitated with NaOAc (0.3M) and 2 volumes of EtOH. Next, thepellet is resuspended at approximately 1 mg/ml in DEPC-treated distilledand deionized water.

Transcription is carried out in a buffer composed of 400 mM Tris HCl (pH8.0), 80 mM MgCl₂, 50 mM DTT and 40 mM spermidine. The subsequentreagents are added in order to one volume of DEPC-treated water at roomtemperature: 1 volume SP6 RNA polymerase transcription buffer; rATP,rCTP and rUTP to 1 mM concentration; rGTP to 0.5 mM concentration;7meG(5′)ppp(5′)G cap analog (New England Biolabs, Beverly, Mass., 01951)to 0.5 mM concentration; the linearized DNA template prepared above to0.5 mg/ml concentration; RNAs in (Promega, Madison, Wis.) to 2000 U/mlconcentration; and SP6 RNA polymerase (Promega, Madison, Wis.) to 3000U/ml concentration. The transcription mixture is incubated for 1 hour at37° C.

The DNA template is then digested by adding 2 U RQ1 DNAse (Promega) permicrogram of DNA template used. The digestion reaction is carried outfor 15 minutes. The transcribed RNA is extracted twice withchloroform/phenol and twice with chloroform. The supernatant solution isprecipitated with 0.3M NaOAc in 2 volumes of EtOH and the pellet isresuspended in 100 ml DEPC-treated deionized water per 500 mltranscription product. Finally, the supernatant solution is passed overan RNAse-free Sephadex G50 column (Boehringer Mannheim # 100 411). Theresultant mRNA is sufficiently pure to be used crystallization.

Example 37

DNA Crystallization

Materials:

A—Purified plasmid DNA (Example 35)

B—spermine

C—MPD (2-Methyl-2,4-Pentanediol)

D—5mM Ca Acetate buffer pH 7.0

E—Deionized water

Procedure:

The amplified DNA (Example 35) is removed from the plasmid byrestriction digestion. The inserted gene is purified from the plasmidvehicle by agarose gel electrophoresis and extraction of the gene ofinterest from the gel band of the appropriate molecular weight.

Using the hanging drop technique, DNA at 5 mg/ml in 5 mM Ca Acetate/20%MPD/1 mM spermine buffer at pH 7.0 is incubated at room temperatureuntil 90% of the DNA has crystallized. The resulting crystals are washedwith crystallization buffer to remove all the soluble material from thecrystals. Then, the crystals are resuspended in fresh crystallizationbuffer to achieve a DNA concentration of 5 mg/ml.

Example 38

RNA Crystallization

Materials:

A—Purified mRNA (Example 36)

B—spermine

C—MPD (2-Methyl-2,4-Pentanediol)

D—5mM Ca Acetate buffer pH 7.0

E—Deionized water

Procedure:

Using the hanging drop technique, RNA (Example 36) at 5 mg/ml in 5 mM CaAcetate/20% MPD/1 mM spermine buffer at pH 7.0 is incubated at roomtemperature until 90% of the RNA has crystallized. The resultingcrystals are washed with crystallization buffer to remove all thesoluble material from the crystals. Then the crystals are resuspended infresh crystallization buffer to achieve RNA concentration of 5 mg/ml.

Example 39

Induction of an Immune Response to HIV gp120 Using DNA Crystals

DNA, coding for HIV gp160, is prepared according to the methods ofExamples 36 and 37. Crystals of HIV gp160 are then used for immunizationof mice. Many genetic clones of both primary and laboratory isolates ofHIV are available from the Aids Research and Reagent Program, NationalInstitutes of Allergy and Infectious Diseases, Rockville Md. 20852, fordesigning vaccines which induce broad neutralizing immunity.

The DNA crystals are maintained in the crystallization buffer and 200μl/mouse is injected into the rear hind leg. The development of animmune response to gp120 is determined by measuring serum antibodies tothe corresponding V3 loop peptide in ELISA on a monthly basis.

Example 40

Induction of an Immune Response to HIV gp120 Using mRNA Crystals

RNA coding HIV gp160 is prepared according to the methods of Examples35, 36 and 38. Crystals of HIV gp160 mRNA are used for the immunizationof mice. Various primary and laboratory isolates of HIV are availablefrom the Aids Research and Reagent Program, National Institutes ofAllergy and Infectious Diseases, Rockville Md. 20852, for designingvaccines which induce broad neutralizing immunity.

The RNA crystals are maintained in the crystallization buffer and 200μl/mouse is injected into the rear hind leg. The development of animmune response to gp120 is determined by measuring serum antibodies tothe corresponding V3 loop peptide in ELISA on a monthly basis.

Example 41

Oligo DNA Crystallization

Materials:

A—Synthetic Oligo DNA

B—spermine

C—MPD (2-Methyl-2,4-Pentanediol)

D—5mM Ca Acetate buffer pH 7.0

E—Deionized water

Procedure:

Using the hanging drop technique, synthetic oligo DNA at 5 mg/ml in 5 mMMg Acetate/30% MPD/1 mM spermine buffer at pH 7.0 is incubated at roomtemperature until 90% of the DNA has crystallized. The resultingcrystals are washed with crystallization buffer to remove all thesoluble material from the crystals. Then, the crystals are resuspendedin fresh crystallization buffer to achieve a DNA concentration of 5mg/ml.

Example 42

Antisense DNA Administration For Inhibition of Gene Expression

Oligo DNA crystals coding for DNA sequences which are complementary tothe sense strand of an mRNA species which is to be suppressed aregenerated as in Example 41. Next, the crystals or a formulationcontaining the crystals is administered to the site where geneexpression is intended to be inhibited. Subsequently, cells will take upthe DNA crystals or dissolved DNA and the oligo DNA and host mRNA willform complementary base pairs and gene expression will be inhibited fora time.

Encapsulated Protein Crystals

Example 43

Large Scale Crystallization of Pseudomonas cepacia Lipase

A slurry of 15 kg crude Pseudomonas cepacia lipase (PS 30 lipase-Amano)(“LPS”) was dissolved in 100 L distilled deionized water and the volumebrought to 200 L with additional distilled deionized water. Thesuspension was mixed in an Air Drive Lightning mixer for 2 hours at roomtemperature and then filtered through a 0.5 μm filter to remove celite.The mixture was then ultrafiltered and concentrated to 10 L (121.4 g)using a 3K hollow fiber filter membrane cartridge. Solid calcium acetatewas added to a concentration of 20 mM Ca(CH₃COO)₂. The pH was adjustedto 5.5 with concentrated acetic acid, as necessary. The mixture washeated to and maintained at a temperature of 30° C. Magnesium sulfatewas added to a 0.2 M concentration, followed by glucopon to a 1%concentration. Isopropanol was then added to a final concentration of23%. The resulting solution was mixed for 30 minutes at 30° C., thencooled from 30° C. to 12° C. over a 2 hour period. Crystallization wasthen allowed to proceed for 16 hours.

The crystals were allowed to settle and soluble protein was removedusing a peristaltic pump with tygon tubing having a 10 ml pipette at itsend. Fresh crystallization solution (23% isopropyl alcohol, 0.2 M MgSO₄,1% glucopon, 20 mM Ca(CH₃COO)₂, pH 5.5) was added to bring theconcentration of protein to 30 mg/ml (O.D. 280 of a 1 mg/mlsolution=1.0, measured using a spectrophotometer at wavelength 280). Thecrystal yield was about 120 grams.

Crosslinked LPS Crystals

Crosslinked Pseudomonas cepacia lipase crystals, sold under the nameChiroCLEC-PC™, are available from Altus Biologics, Inc. (Cambridge,Mass.) were used to produce formulations according to Example 48.Alternatively, lipase crystals as prepared above may be crosslinkedusing any conventional method.

Example 44

Crosslinking of Glucose Oxidase Crystals

We then crosslinked the glucose oxidase crystals prepared in Example 21as follows. The crosslinking procedure involved glutaraldehyde, orglutaraldehyde pretreated with either Tris buffer(2-amino-2-hydroxymethyl-1,3-propanediol), lysine or diaminooctane.

The crosslinking was performed using 60 mg of protein. Tris pretreatedglutaraldehyde (48 mg Tris-base/g glutaraldehyde) was added toconcentrations of 0.2 and 0.6 g/g GO crystals suspended in 0.2 M sodiumphosphate at pH 7.0. Reactions were allowed to proceed for two hours atroom temperature. After two hours, the crystals were filtered and washedover glass fiber paper.

The crosslinked crystals were encapsulated as described in Example 48.No differences were encountered in the encapsulation process between thevariously crosslinked crystals. A representative sample is shown in FIG.21.

Example 45

Crosslinked Candida Rugosa Lipase Crystals

Crosslinked Candida rugosa lipase crystals, sold under the nameChiroCLEC-CR™, are available from Altus Biologics, Inc. (Cambridge,Mass.) and were used to produce formulations according to Example 48.Alternatively, lipase crystals as prepared above, may be crosslinkedusing any conventional method.

Example 46

Crosslinking of Human Serum Albumin Crystals

Crosslinking was performed on the human serum albumin crystals preparedin Example 10. The crosslinking reaction was performed at 4° C. in astirred solution of crystals in the mother liquor containing 50%saturated ammonium sulfate. The crystals were not washed prior tocrosslinking with borate-pretreated glutaraldehyde.

Pretreated glutaraldehyde was prepared by adding one volume of 50%glutaraldehyde (“GA”) to an equal volume of 300 mM sodium borate at pH9. The glutaraldehyde solution was then incubated at 60° C. for 1 hour.The solution was cooled to room temperature and the pH was adjusted to5.5 with concentrated HCl. Next, the solution was rapidly cooled on iceto 4° C.

The pretreated glutaraldehyde (25%) was added to the crystallizationsolution in a stepwise fashion, using 0.05% increments (totalconcentration) at 15 minute intervals to a concentration of 2%. Aliquotsof the crystallization solution used ranged between 1 ml and 500 mlvolume. The crystals were then brought to 5% GA and incubated at 4° C.for 4 hours to allow crosslinking. Finally, albumin crosslinked crystalswere collected by low speed centrifugation and washed repeatedly with pH7.5, 100 mM Tris HCl. Washing was stopped when the crosslinked crystalscould be centrifuged at high speed without aggregation.

Example 47

Crosslinking of Penicillin Acylase

Pretreated glutaraldehyde was prepared by the method of Example 46.

The pretreated glutaraldehyde (25%) was added to the crystallizationsolution in a stepwise fashion, using 0.05% increments (totalconcentration) at 15 minute intervals to a concentration of 1.5%.Aliquots of the crystallization solution used ranged between 1 ml and500 ml volume. Finally, crosslinked crystals were collected by low speedcentrifugation and washed repeatedly with pH 7.5, 100 mM Tris HCl.Washing was stopped when the crosslinked crystals could be centrifugedat high speed without aggregation.

Example 48

Microencapsulation of Protein Crystals in Polylactic-co-glycolic Acid(PLGA)

A. Glycoproteins, Proteins, Enzymes Hormones, Antibodies and Peptides

Microencapsulation was performed using uncrosslinked crystals of lipasefrom Candida rugosa and Pseudomonas cepacia, glucose oxidase fromAspergillus niger and Penicillin acylase from Escherichia coli. Further,microencapsulation was performed using crosslinked enzyme crystals oflipase from Candida rugosa, glucose oxidase from Aspergillus niger andPenicillin acylase from Escherichia coli. Table 13 shows the approximateaverage diameters of samples of the microspheres which were produced bythis example. In addition, human serum albumin or any other proteincrystals or protein crystal formulation produced may be encapsulated bythis technique.

TABLE 13 Microspheres Produced Crosslinked Crosslinked Crystals CrystalsMicrospheres Diameter μm Diameter μm Candida rugosa lipase 90 90 Glucoseoxidase 50 50 Penicillin acylase 90 70 Lipase from Pseudomonas 60cepacia (slurry)

B. Preparation of Dry Crystals:

Crystals or crystal formulations dried according to Example 6 may eachbe used to produce the microspheres of this invention. One process fordrying protein crystals for use in this invention involves air drying.

Approximately 500 mg each of Candida rugosa lipase crystals from Example1 (uncrosslinked and crosslinked), glucose oxidase from Examples 21 and44 (uncrosslinked and crosslinked) and Penicillin acylase from Examples14 and 47 (uncrosslinked and crosslinked) were air dried. First, themother liquor was removed by centrifugation at 3000 rpm for 5 minutes.Next, the crystals were at 25° C. in the fume hood for two days.

C. Polymer and Solvents

The polymer used to encapsulate the protein crystals was PLGA. PLGA waspurchased as 50/50 Poly(DL-lactide-co-glycolide) from BirminghamPolymers, Inc. from Lot No. D97188. This lot had an inherent viscosityof 0.44 dl/g in HFIP@ 30° C.

The methylene chloride was spectroscopic grade and was purchased fromAldrich Chemical Co. Milwaukee, Wis. The poly vinyl alcohol waspurchased from Aldrich Chemical Co. Milwaukee, Wis.

D. Encapsulation of Crystals in PLGA:

The crystals were encapsulated in PLGA using a double emulsion method.The general process was as follows, either dry protein crystals or aslurry of protein crystals was first added to a polymer solution inmethylene chloride. The crystals were coated with the polymer and becamenascent microspheres. Next, the polymer in organic solvent solution wastransferred to a much larger volume of an aqueous solution containing asurface active agent. As a result, the organic solvent began toevaporate and the polymer hardened. In this example, two successiveaqueous solutions of decreasing concentrations of emulsifier wereemployed for hardening of the polymer coat to form microspheres. Thefollowing procedure was one exemplification of this general process.Those of skill in the art of polymer science will appreciate that manyvariations of the procedure may be employed and the following examplewas not meant to limit the invention.

1.0 Use of Dry Protein Crystals

Dry crystals of crosslinked and uncrosslinked Candida rugosa lipaseproduced according to Example 1, crosslinked and uncrosslinked glucoseoxidase produced according to Examples 21 and 45, crosslinked anduncrosslinked penicillin acylase produced according to Example 14 and47, were weighed into 150 mg samples. The weighed protein crystals werethen added directly into a 15 ml polypropylene centrifuge tube (FisherScientific) containing 2 ml of methylene chloride with PLGA at 0.6 gPLGA/ml solvent. The crystals were added directly to the surface of thesolvent. Next, the tube was throughly mixed by vortexing for 2 minutesat room temperature to completely disperse the protein crystals in thesolvent with PLGA. The crystals were allowed to become completely coatedwith polymer. Further vortexing or agitation may be used to keep thenascent microspheres suspended to allow further coating. The polymer maybe hardened as described in section 3.0.

2.0 Use of a Protein Crystal Slurry

A crystal slurry of Pseudomonas cepacia lipase was produced usingapproximately 50 mg of crystals per 200 μl of mother liquor. The crystalslurry was rapidly injected into a 15 ml polypropylene centrifuge tube(Fisher Scientific) with 2 ml of a solution of methylene chloride andpoly(lactic-co-glycolic acid) at 0.6 g PLGA/ml solvent. The needle wasinserted below the surface of the solvent and injected into thesolution. In this case, 150 mg of total protein, or 600 μl of aqueoussolution, was injected. The injection was made using a plastic syringeLeur-lok (Becton-Dickinson & Company) and through a 22 gauge(Becton-Dickinson & Company) stainless steel needle. Next, the proteincrystal-PLGA slurry was mixed thoroughly by vortexing for 2 minutes atroom temperature. The crystals were allowed to be completely coated withpolymer. Further vortexing or agitation was optionally used to keep thenascent microspheres suspended to allow further coating.

3.0 Hardening the Polymer Coating

A two step process was employed to facilitate the removal of methylenechloride from the liquid polymer coat and allow the polymer to hardenonto the protein crystals. The difference between the steps is that theconcentration of emulsifier was much higher in the first solution andthe volume of the first solution is much smaller than the second.

In the first step, the polymer coated crystal and methylene chloridesuspension was added dropwise to a stirred flask of 180 ml of 6%polyvinyl alcohol (hereinafter “PVA”) in water with 0.5% methylenechloride at room temperature. This solution was mixed rapidly for 1minute.

In step two, the first PVA solution containing the nascent microsphereswas rapidly poured into 2.4 liters of cold (4° C.) distilled water. Thisfinal bath was mixed gently at 4° C. for 1 hr with the surface of thesolution under nitrogen. After 1 hr, the microspheres were filteredusing 0.22 μm filter and washed with 3 liters of distilled watercontaining 0.1% Tween 20 to reduce agglomeration.

Example 49

Production of Encapsulated Crystals

Encapsulated microspheres of Pseudomonas cepacia lipase are prepared byphase separation techniques. The crystalline LPS prepared in Example 43is encapsulated in polylactic-co-glycolic acid (“PLGA”) using a doubleemulsion method. A 700 mg aliquot of protein crystals is injected inmethylene chloride containing PLGA (0.6 g PLGA/ml solvent; 10 ml). Themixture is homogenized for 30 sec at 3,000 rpm, using a homogenizer witha microfine tip. The resulting suspension is transferred to a stirredtank (900 ml) containing 6% poly (vinyl alcohol) (“PVA”) and methylenechloride (4.5 ml). The solution is mixed at 1,000 rpm for 1 min. Themicrospheres in the PVA solution are precipitated by immersion indistilled water, washed and filtered. The microspheres are then washedwith distilled water containing 0.1% Tween, to reduce agglomeration anddried with nitrogen for 2 days at 4° C.

Example 50

A. Protein Content of Microspheres

The total protein content of the microspheres prepared in Example 48 wasmeasured. Triplicate samples of 25 mg of the PLGA/PVA microspheres wereincubated in 1 N sodium hydroxide with mixing for 48 hrs. The proteincontent was then estimated using Bradford's method (M. M. Bradford,Analytical Biochemistry, vol. 72, page 248-254 (1976)) and acommercially available kit from BioRad Laboratories (Hercules, Calif.).The protein containing microspheres were compared to PLGA microsphereswithout any crystals and is shown in Table 14.

TABLE 14 Protein Content of Microspheres Protein (%) Protein (%)Crosslinked Uncrosslinked Microspheres Crystals Crystals Unloaded PLGA 0 Microspheres Candida rugosa lipase 30 20 Glucose oxidase 35 28Penicillin acylase 27 25 Lipase from Pseudomonas 39 cepacia (slurry)

The activity per milligram or specific activity of selected samples fromTable 14 was determined and is shown in Table 15.

TABLE 15 Specific Activity Activity/mg Activity/mg CrosslinkedUncrosslinked Microspheres Crystals Crystals Unloaded PLGA 0 0Microspheres Candida rugosa lipase 413 Penicillin acylase 9.63 Lipasefrom Pseudomonas 1414 cepacia (slurry)

Example 51

Protein Release from Microspheres

The release of protein from the PLGA microspheres prepared in Example 48was measured by placing 50 mg of protein encapsulated PLGA microspheresin microcentrifuge filtration tubes containing 0.22 μm filters. Next,600 μl of release buffer (phosphate buffered saline with 0.02% Tween 20at pH 7.4) was added to the microspheres on the retentate side of thefilter. The tubes were incubated at 37° C. to allow dissolution. Tomeasure the amount of protein released with time, samples were taken atdifferent time intervals. The tube was centrifuged at 3000 rpm for 1minute and the filtrate was removed for protein activity and totalprotein measurements. The microspheres were then resuspended withanother 600 μl of release buffer.

A. Total Protein Released From Microspheres

Table 16 shows that almost 90% of the input protein was recovered in allcases. In addition, the percentage of total Pseudomonas cepacia lipasereleased from the microencapsulated protein crystals was relativelyconstant for approximately 5.7 days or until more than 80% of the inputprotein had been released at a rate of 15.8%/day. This long rapidrelease was followed by eight days with a only 0.6% release per day.

In contrast, Table 16 further shows that Candida rugosa lipase crystalsdisplayed the opposite profile, displaying first a slow release whichwas followed by a rapid release phase. In the first three days, about10% of the protein was released with a shallow slope of 2.4%/day. Fromday 4 to day 14, another 80% of the protein was released in a linearfashion and a slope of 7.5%/day. The release profiles shown in Table 16were obtained at 37° C. and at pH 7.4.

These data illustrate that the encapsulated proteins of this inventionare suitable for biological delivery of therapeutic proteins. Variousrates of delivery can be selected by manipulating the choice of proteincrystal, size of the crystals, crosslinking of the crystals, thehydrophobic and hydrophilic characteristics of the encapsulatingpolymer, the number of encapsulations, dose of microspheres and othereasily controllable variables.

TABLE 16 Protein Release From Microspheres % Input Pseudomonas % InputCandida cepacia Lipase rugosa lipase Time (hr) Released Released 0 0 018 28 2 41 56 5 89 75 10 137 82 22 210 84 34 234 85 47 306 86 70 330 8786

B. Protein Activity Released From Microspheres

The biological activity of the protein released with time was measuredusing the olive oil assay for lipase microspheres. These results areshown in Table 17.

The biological activity of the released protein, as shown in Table 17,demonstrates that the microspheres protect and release active protein.The cumulative percent activity released, calculated based on the amountof input protein, was closely correlated with the total protein released(compare Table 16 and Table 17). The two different crystal lipasesreleased essentially 100% active protein. Even after 7 days of immersionat 37° C., the protein that was released from the microspheres was fullyactive.

TABLE 17 Activity of Released Protein % Input Pseudomonas % InputCandida cepacia Lipase Rugosa lipase Time (hr) Activity ReleasedActivity Released 0 0 0 18 28 2 41 56 5 89 75 10 137 82 22 210 84 34 23485 47 306 86 70 330 87 86

The activity measurements set forth above were made using the olive oilassay described in Example 8.

Example 52

Microscopic Examination of PLGA Microspheres

In order to visualize whether the crystals were intact afterencapsulation, PLGA microspheres prepared according to Example 48 wereexamined under an Olympus BX60 microscope equipped with DXC-970MD 3CCDColor Video Camera with Camera Adapter (CMA D2) with Image ProPlussoftware. Samples of dry microspheres were covered with a glasscoverslip, mounted and examined under 10× magnification, using anOlympus microscope with an Olympus UPLAN F1 objective lens 10×/0.30 PH1(phase contrast), the crystals were readily visualized and the crystalsize determined. Microsphere and crystal sizes were determined usingImage Pro Software from Olympus and 0.5-150 μm sizing beads provided bythe manufacturer. The size of the outer PLGA microspheres wasdetermined, as well as for the crystals.

FIG. 17 depicts crosslinked enzyme crystals of lipase from Candidarugosa encapsulated by the method of Example 48. The crystal size wasapproximately 25 μm and the microspheres were approximately 90 μm. Themagnification was 250×.

FIG. 18 depicts uncrosslinked enzyme crystals of lipase from Candidarugosa encapsulated by the method of Example 48. The crystal size wasapproximately 25 μm and the microspheres were approximately 120 μm. Themagnification was 250×.

FIG. 19 depicts crosslinked enzyme crystals of Penicillin acylase fromEscherichia coli encapsulated by the method of Example 48. The crystalsize was approximately 25 μm and the microspheres were approximately 70μm. The magnification was 250×.

FIG. 20 depicts uncrosslinked enzyme crystals of Penicillin acylase fromEscherichia coli encapsulated by the method of Example 48. The crystalsize was approximately 50 μm and the microspheres were approximately 90μm. The magnification was 250×.

FIG. 21 depicts crosslinked enzyme crystals of glucose oxidase fromAspergillus niger encapsulated by the method of Example 48. The crystalsize ranged from 0.5 to 1 μm and the microspheres were approximately 50μm. The magnification was 500×.

FIG. 22 depicts uncrosslinked enzyme crystals of glucose oxidase fromAspergillus niger encapsulated by the method of Example 48. The crystalsize ranged from 0.5 to 1 μm and the microspheres were approximately 50μm. The magnification was 500×.

FIG. 23 depicts uncrosslinked enzyme crystals of lipase from Pseudomonascepacia, encapsulated as a slurry in the mother liquor by the method ofExample 48. The crystal size was approximately 2.5 μm and themicrospheres were approximately 60 μm. The magnification was 500×.

FIG. 24 depicts uncrosslinked enzyme crystals of lipase from Pseudomonascepacia. The crystal size was approximately 2.5 μm. The magnificationwas 1000×.

Example 53

Protein Release

The release of proteins from the PLGA microspheres is measured byplacing 50 mg of PLGA microspheres in micro-centrifuge filtration tubescontaining 0.22 μm filters. A 600 μl aliquot of release buffer (10 mMHEPES, pH 7.4, 100 mM NaCl, 0.02% Tween, 0.02% azide) is added tosuspend the microspheres on the retentate side of the filter. The tubesare sealed with 3 cc vial stoppers and covered by parafilm. Themicrospheres are then incubated at 37° C. Samples are taken over time bycentrifugation (13,000 rpm, 1 min) of the tubes. The filtrate is removedand the microspheres are resuspended with 600 μl of the release buffer.The quality of the released protein is assayed by SEC-HPLC and enzymaticactivity.

The shape and size of the protein crystals may be chosen to adjust therate of dissolution or other properties of the protein crystalformulations of this invention.

Example 54

Encapsulation of Lipase Crystals Using a Biological Polymer

Biological polymers are also useful for encapsulating protein crystals.The present example demonstrates encapsulation of crosslinked anduncrosslinked crystals of Candida rugosa lipase crystals. Theuncrosslinked and crosslinked crystals were prepared as described inExample 1 and 45. Antibodies and chemicals were purchased from Sigma.

1.0 Preparation of Coated Crystals

A solution of 1.5 ml of bovine serum albumin (“BSA”) at 10 mg/ml wasprepared, in 5 mM phosphate buffer adjusted to pH 7. Next, 15 ml of a 10mg/ml suspension of Candida rugosa lipase crystals was prepared in 5 mMK/Na phosphate buffer, 1 M NaCl, at pH 7 (“buffer”). The BSA solutionwas added to the crystal solution and the two solutions were mixedthoroughly. The crystals were incubated in the BSA for 30 min with slowmixing using an orbital shaker. Following the incubation with BSA, thecrystals were dryed overnight by vacuum filtration. The dryed crystalswere resuspended in buffer without albumin. The crystals were washedwith buffer until no protein could be detected in the wash as measuredby absorbance at280 nm or until the A_(280nm) was <0.01. The crystalswere recovered by low speed centrifugation.

2.0 Detection of the Albumin Coat

The coated crystals were evaluated by Western blotting to confirm thepresence of the albumin layer Following washing, coated protein crystalswere incubated in 100 mM NaOH overnight to dissolve the microspheresinto the constituent proteins. The samples were neutralized, filteredand analyzed by SDS-PAGE immunoblot according to Sambrook et al.“Molecular Cloning: A Laboratory Manual”, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989).

The results of SDS-PAGE immunoblot of both albumin coated crosslinkedand uncrosslinked crystal microspheres of Candida rugosa lipase revealeda single immunoreactive species having the same molecular weight asalbumin.

Samples of the albumin coated crosslinked and uncrosslinked crystalmicrospheres of Candida rugosa lipase were incubated with afluorescene-labeled anti-BSA antibodies which specifically recognize andbind to bovine serum albumin. Next, excess antibody was removed thoroughwashing with phosphate buffer. Microscopic examination of thesefluorescently labeled albumin coated crystal microspheres under afluorscent microscope revealed specific fluorescene-labeling of themicrospheres. Uncoated lipase crystals were used as control and theseshowed no specific binding of the antibody.

While we have hereinbefore described a number of embodiments of thisinvention, it is apparent that our basic constructions can be altered toprovide other embodiments which utilize the processes and compositionsof this invention. Therefore, it will be appreciated that the scope ofthis invention is to be defined by the claims appended hereto ratherthan by the specific embodiments which have been presented hereinbeforeby way of example.

We claim:
 1. A formulation comprising: a) a batch-processed proteincrystal, wherein said protein is selected from the group consisting ofhydrolases, lipases, bacterial proteins and human growth hormone; and b)an excipient, wherein said excipierit and said protein crystal are in aratio between 0.01:99.99 and 30:70 (W/W); wherein said formulation hasat least a 59 fold greater shelf life when stored at 40° C. and 75%humidity than a nonformulated form of said protein crystal when storedat 40° C. and 75% humidity, as measured by the time required for thespecific activity of the protein to decrease by 50% (T_(½)).
 2. Aformulation comprising: a) a batch-processed protein crystal, whereinsaid protein is an oxidase enzyme; and b) an excipient, wherein saidexcipient and said protein crystal are in a ratio between 0.01:99.99 and30:70 (W/W); wherein said formulation has at least a 59 fold greatershelf life when stored at 40° C. and 75% humidity than a nonformulatedform of said protein crystal when stored at 40° C. and 75% humidity, asmeasured by the time required for the specific activity of the proteinto decrease by 50% (T_(½)).
 3. A formulation comprising: a) abatch-processed protein crystal, wherein said protein is human Serumalbumin; and b) an excipient, wherein said excipient and said proteincrystal are in a ratio between 0.01:99.99 and 30:70 (W/W); wherein saidformulation has at least a 59 fold greater shelf life when stored at 40°C. and 75% humidity than a nonformulated form of said protein crystalwhen stored at 40° C. and 75% humidity, as measured by the time requiredfor the specific activity of the protein to decrease by 50% (T_(½)). 4.A formulation comprising: a) a batch-processed protein crystal, whereinsaid protein is selected from the group consisting of hydrolases,lipases, bacterial proteins and human growth hormone; and b) anexcipient, wherein said excipient and said protein crystal are in aratio between 0.01:99.9 and 30:70 (W/W); wherein said formulation has atleast a 60% greater shelf life when stored at 50° C. than anonformulated form of said protein crystal when stored at 50° C., asmeasured by the time required for the specific activity of the proteinto decrease by 50% (T_(½)).
 5. A formulation comprising: a) abatch-processed protein crystal, wherein said protein is an oxidaseenzyme; and b) an excipient, wherein said excipient and said proteincrystal are in a ratio between 0.01:9.99 and 30:70 (W/W); wherein saidformulation has at least a 60% greater shelf life when stored at 50° C.than a nonformulated form of said protein crystal when stored at 50° C.,as measured by the time required for the specific activity of theprotein to decrease by 50% (T_(½)).
 6. A formulation comprising: a) abatch-processed protein Crystal, wherein said protein is human serumalbumin; and b) an excipient, wherein said excipient and said proteincrystal are in a ratio between 0.01:99.99 and 30:70 (W/W); wherein saidformulation has at least a 60% greater shelf life when stored at 50° C.than a nonformulated form of said protein crystal when stored at 50° C.,as measured by the time required for the specific activity of theprotein to decrease by 50% (T_(½)).
 7. A formulation comprising: a) abatch-processed protein crystal, wherein said protein is selected fromthe group consisting of hydrolases, lipases, bacterial proteins andhuman growth hormone; and b) an excipient, wherein said excipient andsaid protein crystal are in a ratio between 0.01:99.99 and 30:70 (W/W);wherein said formulation loses less than 20% α-helical structuralcontent of the protein after storage for 4 days at 50° C., wherein asoluble form of said protein loses more than 50% of its α-helicalstructural content after storage for 6 hours at 50° C., as measured byfourier transform infrared (FTIR).
 8. A formulation comprising: a) abatch-processed protein crystal, wherein said protein is an oxidaseenzyme; and b) an excipient, wherein said excipient and said proteincrystal are in a ratio between 0.01:99.99 and 30:70 (W/W); wherein saidformulation loses less than 20% α-helical structural content of theprotein after storage for 4 days at 50° C., wherein a soluble form ofsaid protein loses more than 50% of its α-helical structural contentafter storage for 6 hours at 50° C., as measured by fourier transforminfrared (FTIR).
 9. A formulation comprising: a) a batch-processedprotein crystal, wherein said protein is human serum albumin; and b) anexcipient, wherein said excipient and said protein crystal are in aratio between 0.01:99.99 and 30:70 (W/W); wherein said formulation losesless than 20% α-helical structural content of the protein after storagefor 4 days at 50° C., wherein a soluble form of said protein loses morethan 50% of its α-helical structural content after storage for 6 hoursat 50° C., as measured by fourier transform infrared (FTIR).
 10. Theformulation according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8 or 9,wherein said protein is greater than 10,000 Daltons in molecular weight.11. The formulation according to any one of claims 1, 2, 3, 4, 5, 6, 7,8 or 9, wherein said excipient is selected from the group consisting ofsucrose, trehalose, lactitol, gelatin, hydroxypropyl-β-cyclodextrtn,methoxypolyethylene glycol and polyethylene glycol.
 12. A microspherecomprising the formulation according to any one of claims 1, 2, 3, 4, 5,6, 7, 8 or
 9. 13. The formulation according to any one of claims 1, 4 or7, wherein said ratio of said excipient to said protein crystal isbetween 0.1:99.9 and 10:90 (W/W).
 14. The formulation according to claim13, wherein said ratio of said excipient to said protein crystal isbetween 1:99 and 10:90 (W/W).
 15. The formulation according to accordingto any one of claims 1, 4 or 7, wherein said lipase is Candida rugosalipase.
 16. The formulation according to according to any one of claims1, 4 or 7, wherein said hydrolase is penicillin acylase.
 17. Theformulation according to any one of claims 1, 4 or 7, wherein saidlipase is Pseudomonas cepacia lipase.
 18. The formulation according toaccording to any one of claims 2, 5 or 8, wherein said oxidase enzyme isglucose oxidase.
 19. The formulation according to any one of claims 2, 5or 8, wherein said ratio of said excipient to said protein crystal isbetween 0.1:99.9 and 10:90 (W/W).
 20. The formulation according to claim19, wherein said ratio of said excipient to said protein crystal isbetween 1:99 and 10:90 (W/W).
 21. The formulation according to any oneof claims 3, 6 or 9, wherein said ratio of said excipient to saidprotein crystal is between 0.1:99.9 and 10:90 (W/W).
 22. The formulationaccording to claim 21, wherein said ratio of said excipient to saidprotein crystal is between 1:99 and 10:90 (W/W).